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Two populations of tyrosine hydroxylase-positive cells occur in the spinal cord of the chick embryo and hatchling
I.ctter~, 83 (I 987) 253 25S t-lsex icr Scicntitic Publishers lrehmd l_.td.
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Two populations of tyrosine hydroxylase-positive cells occur in the spinal cord of the chick embryo and hatchling .lames A. Wallace, Richard M. Mondragon, Permelia C. A l l g o o d . Thomas J. 1loffman and Rolanda R. Maez lIcparlmc.t
~;I -I n u l o m v. ~ ' n i v c r s i t y ~,I ,%'u M c ~ . r , ,~,'~ho,d ol .'~tcdicim'. ,.I It, u q u e r q m ' ,
N..~,I ,~ 7 I.? I : I ,5,'. -I
[l~.t.t_t.I ed -...hint 1087; Re~.ised ~crsion received 24 August I'-)8;7: Accepted :"; Scplember 10~71 hcL ~ , . d r
Neurotransmilter phcnotype development: Tyrosine-hydrox',lase (I11)-contammg cell: AnliT I I immunc,cytochenfistr?,: ('alecholamme; Spinal cord deveh~pment: ('hick cmbr_,,~
The existence of lyrosme hydroxylase ITll)-contaming neurons in the spinal cord of the chink embryo ssas investigated b+ anti-TH immunocytochemistr). "fwo populations of intensely immunostained cells were observed ahmg the entire exlent of the cord. beginning tale in chick embryogenesis. One group of fl-i-positi,.e cells was particularly numerous and found ventral to, the central canal. The other grmtp. which ~ a s smaller in number, v, as located ~,long the superlicial and lateral border of the dorsal horn of the spinal c~rd. When examined by the glyoxylic acid histolluorescence technique, cells could be visuali/ed onl~ xer.,,' infrequently ventral to the central canal, and not al all v, ithin the dorsal horn. t lowever, after pretreatmcnt of hatchlings v, ith the catecholamine synthesis precursor I.-DOPA. cells ventral l~ Ihe canal ,acre rcadil?, obscr,,ed b3, histofluoresccnce, while the dorsally located cells were seldom xisualb.cd Since these populations of T | l-positive cells appear to only partially express the catechokuninergic phcnot?,p¢. Ihese cells ma.', p r m idea model in which factors regulating the expression of neurotransmitter phenoL,, pc,, can be cxamined in neurons of the de,.eloping ('NS.
Besides the descending catecholaminergic (CA) innervation of the spinal cord from brainstem CA nuclei [I, 23], thcre is growing evidence of CA cell groups intrinsic to the spinal cord, itself, not only in lower vertebrate species [14. 15, 17, 241, but also in the rat [3, 12, 181. Several developmental studies have reported the presence of spinal cord cells which express some characteristics of the CA transmitter phenotype. Fhcse cells were observed either by CA histofluorescence or by the immunocvtochemical detection of CA biosynthetic enzymes [2.6, 17, 19, 20]. In several investigations, the presence of these spinal cord cells was described as being transient [2. 6. I 8, 20], which raises the issue of how the expression of neurotransmitter phenot,\,pes is regulated in the developing central nervous system (('NS). The present investigation examined the occurrencc of cells that express one component of the CA transmitter phenot2,pe in the spinal cord of the chick embryo [22]. These cells were detected inmmnocylochemically by their content of tyrosine hydrox.I.A. Wallace, l)eparlment of ,,\naton~.~, tJni~.ersitk of N e ~ Mexico School of Mcdtcinc. .,\lbuquCrquc. N I',,I ,~7131. T,'.SA. ('orrcv~ondcmc
254 ylase (TH), the first enzyme in the pathway of CA biosynthesis. Chick embryos were obtained at the ages of 9, 12, 15, 17 and 21 days of incubation (E21) and were staged [7] at the time of sacrifice. Embryos were perfused with 4% paraformaldehyde and representative portions of the spinal cord from cervical, thoracic, lumbar and sacral levels were processed for paraffin embedding and immunocytochemistry, as described in detail in a previous publication [2 I]. Sections were cut at 9 pm thickness in a plane transverse to the long axis of the cord. The sections were stained immunocytochemically by the peroxidase avidin biotin complex (ABC) procedure of Hsu et al. [8]. The antiserum used in this study (diluted at 1/400 to 1/600) was obtained commercially (Eugene Tech International), and was raised in rabbits against TH isolated from bovine adrenals. The antiserum to TH produced in this manner has been characterized previously [16]. In adult and developing chickens, the anti-TH immunoperoxidase staining patterns matched the locations of cells visualized by the CA histofluorescence in the central and peripheral nervous systems [4, 5, 9]. The earliest detection of immunostaining within perikarya of the spinal cord was at El5. By far the most frequent of these were cells situated ventral to the central canal, or occasionally located in the lateral walls of the canal, along the entire length of the cord. In the majority of cases, these small, lightly stained TH-positive perikarya were found within the ependymal layer o f cells surrounding the central canal, or just outside the boundary of the ependymal cell layer. Stained processes emanating from these cells were seldom seen. Also at El 5, another group ofimmunostained cells was observed for the first time within the dorsal horn of the spinal cord. However, these cells were encountered only very infrequently. They were also lightly stained and had few elongated processes. By E17, these two groups of TH-positive cells appeared considerably more mature. Both sets of cells not only stained more intensely, they also possessed more elaborate cellular processes. In addition, the population of cells ventral to the central canal also seemed more numerous (with commonly 5 or more cells observed per section) and were larger in size (8--.10 pm in diameter). The majority of the ventrally located cells had thick, well-stained processes that contacted the lumen of the central canal. Furthermore, a large number of these cells also possessed fine processes which originated from the side of the cells away from the canal. These processes were axon-likc in that they appeared varicose, yet they could only be followed over short distances. At EI 7, the number of cells found in the dorsal horn of the spinal cord increased dramatically over the few cells observed at E 15. Often one or more of these cells were observed per section at all levels of the cord. A striking feature of the anti-TH stained cells in the dorsal horn was that their size was often comparable to that of large motoneurons (both ranging from 20 to 25 pm in diameter). These large immunostained cells were frequently situated along the perimeter of the dorsal horn, especially near its boundary with the white matter of the lateral funiculus (Fig. 1A). Occasionally, stained cells were also found deeper within the dorsal horn near the preganglionic sympathetic cell column (nucleus of Terni) which is bilateral and dorsolateral to the central canal in thoracic and upper lumbar cord segments. At hatching (E21), the anti-TH-immunostained cells of the dorsal horn varied considerably in their morphology. While some appeared as spindle-shaped cells, others were pyrami-
255
dal or multipolar (Fig. I B), with several well-stained and variously branched processes. Few differences were noted in the appearance of immunostained cells ventral to the central canal between El7 and E21. C o m m o n to both ages, the processes that .
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Fig. 1. A: schematic diagram of a cross-section of the spinal cord at the junction of the lower thoracic and upper lumbar cord segment, l~[i, location of anti-T|l immunostained cells (dots) in the spinal cord of chick embryos at 17 days of incubation (El 7). The cells are situated along the perimeter of the dorsal horn adjacent to the lateral funiculus, near the nucleus of Terni (T), and ventral to the central canal (col. ( l h i s diagram represents a composite of approximately 6---8 sections (at 9/tin) to show the various positions of the dorsally located cells, not their relative density within a single section, llowever, for the cells shown ventral to the central canal, the number of cells illustrated is representative of their frequency that can be encountered in a single section.) Right. cytoarchitectonic organization of laminae within the lumbar cord of the chicken [13]. B: deeply-stained ceils in the dorsal horn at E21 appear pyramidal as well as multipolar, with easily visualized elaborate and branched processes. The cells shown in this tigure were from a section taken slightly oblique to the saginal plane of the cord due to unequal shrinkage of the face of the paralfin-embedded tissue. C': ventral to the central canal (*), several immunostained cells are seen. some of which retain their contact with the canal. However, as typified at E21 (the age shown m both (" and D), an increasing number of Tll-positive cells are also observed to have lost their contact with the central canal and are located more ventrally, further away from the canal. D: upon closer examination, the thick processes of the cells located immediately ventral to the canal have large club-like endings (arrow) within the lumen of the canal. Bars in B. (" = 25 itm: in D = I0 ltm.
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projected to the central canal ended in enlarged club-like swellings within the lume~ of the canal (Fig. I D). Howevcr, at E21 compared to El7, a greater number of the cells had lost contact with the lumen and were tbund more ventrally away from the canal, often considerably outside the ependymal cell layer (Fig. I C). The locations of TH-positive cells reported in this investigation correlate well wit h the description of CA neurons occurring within the spinal cords o1" other species. In particular, the immunostained cells vcntral to the central canal in the chick embryo appear analogous to cells demonstrated by CA histofluorcscence or by tmti-Tll immunocytochemistry in several lower vcrtebrate species [14, 15, 17, 24]. In contrast. the cells within the dorsal horn, described here, have not been observed in lower species, but have been found in almost identical locations by CA histofluorescence and anti-TH immunocytochemistry in the rat [3, 18]. Thus, it appears that the chicken is intermediate along the evolutionary line with respect to the occurrence and position o f these CA cells intrinsic to the spinal cord. Since catecholamines have been demonstrated histochemically within cells found ventral to the central canal in lower vertebrate species, as well as within cells of the dorsal horn of the spinal cord in higher vertebrate species, we examined whether the two populations of TH-positive cells in the chick spinal cord also possess CA. in these studies, we investigated mid-cervical and upper lumbar cord segments in chick
F i g 2. A: fluorescence micrograph of the upper lumbar spinal cord o f a hatchling (6 12 h old) demonstrating glyoxylic acid histofluorescence of catecholamines ICA) in the vicinity of the central canal (*) m a control animal without drug treatment. Note the large number of brightly fluorescent terminals surrounding the ependymal cells lining the central canal, but the absence of fluorescence within cells ventral to the canal where numerous "Ill-positive perikarya occur. B: at the same level of the cord in a hatchling treated with 300 mg/kg nialamide and 300 mg/kg L-DOPA (4 h prior to sacrifice), numerous CA-positive cells appear ventral to the canal in almosl every section On the same location as TH-immunostained cells observed at this age~. These CA-histofluore~ent cells possess thick-proces,~s (arrow) projecting towards anti contacting the lumen of the central canal. Bar 25 ilm.
25,'7
hatchlings (within 6 12 h of hatching). Utilizing the glyoxylic acid histofluorescencc technique (as described by Kimura et al. [I I]), no fluorescent cells could be visualized at either level of the cord in most control animals (Fig. 2A). (However, in a small number of cases, a few weakly fluorescent cells were found at both cord levels. These few CA-positive cells were located ventral to the central canal, but never in the dorsal horn.) In contrast, after pretreating chick hatchlings with a monoamine oxidase inhibitor (either nialamide or pargyline, at 150 300 mg..'kg, i.p.) in combination with the ('A synthesis precursor. L-I)OPA (l.-fl-3,4-dihydroxyphenylalaninc, at 150 300 m g k g , i.p.) for 4 h prior to sacrifice, far greater numbers of cells ventral to the canal were observed with brighter fluorescence (Fig. 2B) at both cord levels. After the same pharmacologic treatments, however, only a few cells in the dorsal horn were CApositive. These involved only those cells located ventrally near the nucleus of Terni. rather than within regions of the dorsal horn where the majority of TH-positivc cells were located. The conversion of the non-fluorescent compound. I.-DOPA, to a fluorescent ('A product, suggests that the TH-positive cells ventral to the central canal in the chick may also possess aromatic L-amino acid decarboxylase (AAI)C). the en~,yme that converts I - I ) O P A to the fluorescent product, dopamine. There are other reports of TH-positive neurons within the CNS that do not concomitantly contain detectable levels of catecholamines, nor A A D C [6, 10]. Furthermore, in the one case examined [I 0]. these TH-imrnunostained cells could not convert t.-DOPA to a fluorescent compound. Moreover, the locations of the cells mentioned in these previous studies (within the ventrolateral portions of the ventral horn of the spinal cord in the embryonic rat [6], and within the inferior colliculus of the postnatal rat pup [10]) do not have an ex.olutionary history of possessing catecholamines. In contrast, the FI lstained cells ventral to the central canal in the chick are comparable in location to dopamine-containing spinal cord cells in lower vertebrate species [14]. In a further cvohttionary context, the dorsally-situated TH-positive cells in the chick spinal cord are also of interest because they appear in higher vertebrate species [3. 18], rather than in lower vertebrate species. Overall, both populations of spinal cord TH-irnnlunoreactivc cells in the chick may represent a model in which to study factors rcgt.lating the expression of the ('A neurotransmitter phenotype in the developing CNS. The authors would like to thank Dr. William I)ail for his review of this article and to Susan H o m e r for her help in preparing the manuscript. This research was supported by NSF Grant BNS-8511079, N I H Grant NS-20039 and Minority' Biomedical Research Support Grant RR-08139.
I (.'arlsson. A., Fal,.:k, B.. Fuxe, K. and llillarp, N.-A., Cellular Iocalizalion of monoamines in the spinal cord. Acta. PhysioI. S c a n d , 60(196~,) 112 119. 2 ('ommissiong, J.W., The development of catecholaminergi,: nerves in lhe spinal cord of rail. I 1. Regional development, I)ev. Brain Res.. I I (1983) 75.92. 3 l)ictl, M., Arluison. M.. Mouchet. P., Feuerstein. C., Manier. M. and Thibault, J., Immunohistochcmical demonstraticm of calecholaminergie cell bodies in the spinal cord of the rat, Hislochemistr,.. ~2 ([~JRYJYg5 3gg~.
258 4 DubS, k and Parent. A., The monoamine-.containing neurons in avian brain. I. A study of the brain stem of the chicken (Gallus domesticus) by means of fluorescence and acetylcholinesterase histochemistry, J. ('omp. Neurol., 196 (1981) 695 708. 5 Enemar, A.. Falck. B. and Hakanson, R., Observations on the appearance of norepinephrine in the sympathetic nervous system of the chick embryo. Dev. Biol., I 1 (1965) 268-283. 6 Foster, G.A., Schultzberg, M., Dahl, D., (Joldstein, M. and Verhofstad, A.A.J., Ephemeral existence of a single catecholamine synthetic enzyme in the olfactory placode and the spinal cord of the embryonic rat, Int. J. Dev. Neurosci., 3 (1985) 597-608. 7 Hamburger, V. and Hamilton, H.U. A series o1"normal stages in the development of the chick embryo. J. Morph.. 88 (1951) 49 92 8 1-1su,S.M., Raine, L. and Fanger, I1., Use of avidin-biotin-peroxidasecomplex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem.. 29 ( 1981 ) 577 580 9 Ikeda, tl. and Gotoh, J., Distribution of monoamine-containing cells in the central nervous system of the chicken, Jpn. J. Pharmacol., 21 (1971) 763-784. 10 Jaeger. C.B. and Joh, "I".14..Transient expression of tyrosine hydroxylase in some neurons of the developing inferior colliculus of the rat, I)e,,. Brain Res.. I I (1983) 128-132. I 1 Kimura. [t., Nagai, T., hnamoto, K. and Maeda, T.. A sensitive wet-histofluorescence method by glyoxylic acid for central catecholamine terminals, Acta Histochem. Cytochem., 12 (I 979) 57 I. 12 Mariani, A.P., Caserta, M.T. and Barker, J.l,.. Morphological and histochemical characterization of three types of dopamine-containing neurons in primary cultures of mouse and rat spinal cord, Brain Res., 376 (1986) 335 341. 13 Martin. A.H., A cytoarchitectonic scheme for the spinal cord of the domestic fowl, Gallus gallus dome.~titus: lumbar region, Acta Morphol. Neerl.-Scand., 17 (1979) 105 117. 14 Ochi. J.. Yamamoto, T. and Hosoya, Y., Comparative study of the monoamine neuron system in the spinal cord of the lamprey and hagfish. Arch. Histol. Jap., 42 (1979) 327 336. 15 Parent, A. and Northcutt, R.(J., The monoamine-containing neurons in the brain of the garfish, Lepi,~,steus osseus. Brain Res. Bull.. 9(1982) 189 204. 16 Pickel, V.M., Joh. TH.. Field, P.M., Becker, ('.(;. and Reis, D.J., Cellular localization of tyrosine hydroxylase by immunocytochemistry, J. Histochem Cytochem., 23 (1975) I-12. 17 Sims. T.J., The development of monoamine-containing neurons in the brain and spinal cord of the salamander, .4mh.vstorna mexicanum. J. Comp. Neurol., 173 (1977) 319 336. 18 Singhaniyom, W., Wreford, N.G.M. and Guldner, F.-H., Asymmetric distribution of catecholaminecontaining neuronal perikarya in the upper cervical spinal cord of rat, Neurosci. Lctt., 41 (1983) 91 97. 19 Tcitelman, (i., Gershon, M.D., Rothman, T.P., Joh, T.H. and Reis, D.J., Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats. Dev. Biol., 86(1981) 348 355. 20 Feitclman, O.. Jaeger, C.B., Albert, V., Joh, T.H. and Reis, D.J., Expression of amino acid dccarboxylast ill proliferating cells of the neural tube and notochord of developing rat embryo. J. Ncurosci., 3(1983) 1379 138g 21 Wallace, J.A., An immunocytochemical study of the development of central serotoninergic neurons in the chick embryo, J. Comp. Ncurol., 236 (1985) 443 453. 22 Wallace, J.A., Mondragon, R.M., Allgood, P.C., I-loffman, T.J. and Maez, R.R., The immunocytochcmic;,l detection of tyrosine hydroxylase-containing ceils in the spinal cord of the chick embryo, Anat. Rec., 214(1986) 140A. 23 Westlund, K.N.. Bowker, R.M., Ziegler, M.G. and Coulter, ,I.D., Descending noradrenergic projections and their spinal terminations. In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 219-238. 24 Wolters, J.O., TenDonkelaar, H.J. and Verhofstad, A.A.J., Distribution of catecholamines in the brainstem and spinal cord of the lizard Varanus exanthematicus." an immunohistochemical study based on the use of antibodies to tyrosine hydroxylase, Neuroscience, 13 (1984)469-493.
253
:Vcuro.wwme
NSL o5o23
Two populations of tyrosine hydroxylase-positive cells occur in the spinal cord of the chick embryo and hatchling .lames A. Wallace, Richard M. Mondragon, Permelia C. A l l g o o d . Thomas J. 1loffman and Rolanda R. Maez lIcparlmc.t
~;I -I n u l o m v. ~ ' n i v c r s i t y ~,I ,%'u M c ~ . r , ,~,'~ho,d ol .'~tcdicim'. ,.I It, u q u e r q m ' ,
N..~,I ,~ 7 I.? I : I ,5,'. -I
[l~.t.t_t.I ed -...hint 1087; Re~.ised ~crsion received 24 August I'-)8;7: Accepted :"; Scplember 10~71 hcL ~ , . d r
Neurotransmilter phcnotype development: Tyrosine-hydrox',lase (I11)-contammg cell: AnliT I I immunc,cytochenfistr?,: ('alecholamme; Spinal cord deveh~pment: ('hick cmbr_,,~
The existence of lyrosme hydroxylase ITll)-contaming neurons in the spinal cord of the chink embryo ssas investigated b+ anti-TH immunocytochemistr). "fwo populations of intensely immunostained cells were observed ahmg the entire exlent of the cord. beginning tale in chick embryogenesis. One group of fl-i-positi,.e cells was particularly numerous and found ventral to, the central canal. The other grmtp. which ~ a s smaller in number, v, as located ~,long the superlicial and lateral border of the dorsal horn of the spinal c~rd. When examined by the glyoxylic acid histolluorescence technique, cells could be visuali/ed onl~ xer.,,' infrequently ventral to the central canal, and not al all v, ithin the dorsal horn. t lowever, after pretreatmcnt of hatchlings v, ith the catecholamine synthesis precursor I.-DOPA. cells ventral l~ Ihe canal ,acre rcadil?, obscr,,ed b3, histofluoresccnce, while the dorsally located cells were seldom xisualb.cd Since these populations of T | l-positive cells appear to only partially express the catechokuninergic phcnot?,p¢. Ihese cells ma.', p r m idea model in which factors regulating the expression of neurotransmitter phenoL,, pc,, can be cxamined in neurons of the de,.eloping ('NS.
Besides the descending catecholaminergic (CA) innervation of the spinal cord from brainstem CA nuclei [I, 23], thcre is growing evidence of CA cell groups intrinsic to the spinal cord, itself, not only in lower vertebrate species [14. 15, 17, 241, but also in the rat [3, 12, 181. Several developmental studies have reported the presence of spinal cord cells which express some characteristics of the CA transmitter phenotype. Fhcse cells were observed either by CA histofluorescence or by the immunocvtochemical detection of CA biosynthetic enzymes [2.6, 17, 19, 20]. In several investigations, the presence of these spinal cord cells was described as being transient [2. 6. I 8, 20], which raises the issue of how the expression of neurotransmitter phenot,\,pes is regulated in the developing central nervous system (('NS). The present investigation examined the occurrencc of cells that express one component of the CA transmitter phenot2,pe in the spinal cord of the chick embryo [22]. These cells were detected inmmnocylochemically by their content of tyrosine hydrox.I.A. Wallace, l)eparlment of ,,\naton~.~, tJni~.ersitk of N e ~ Mexico School of Mcdtcinc. .,\lbuquCrquc. N I',,I ,~7131. T,'.SA. ('orrcv~ondcmc
254 ylase (TH), the first enzyme in the pathway of CA biosynthesis. Chick embryos were obtained at the ages of 9, 12, 15, 17 and 21 days of incubation (E21) and were staged [7] at the time of sacrifice. Embryos were perfused with 4% paraformaldehyde and representative portions of the spinal cord from cervical, thoracic, lumbar and sacral levels were processed for paraffin embedding and immunocytochemistry, as described in detail in a previous publication [2 I]. Sections were cut at 9 pm thickness in a plane transverse to the long axis of the cord. The sections were stained immunocytochemically by the peroxidase avidin biotin complex (ABC) procedure of Hsu et al. [8]. The antiserum used in this study (diluted at 1/400 to 1/600) was obtained commercially (Eugene Tech International), and was raised in rabbits against TH isolated from bovine adrenals. The antiserum to TH produced in this manner has been characterized previously [16]. In adult and developing chickens, the anti-TH immunoperoxidase staining patterns matched the locations of cells visualized by the CA histofluorescence in the central and peripheral nervous systems [4, 5, 9]. The earliest detection of immunostaining within perikarya of the spinal cord was at El5. By far the most frequent of these were cells situated ventral to the central canal, or occasionally located in the lateral walls of the canal, along the entire length of the cord. In the majority of cases, these small, lightly stained TH-positive perikarya were found within the ependymal layer o f cells surrounding the central canal, or just outside the boundary of the ependymal cell layer. Stained processes emanating from these cells were seldom seen. Also at El 5, another group ofimmunostained cells was observed for the first time within the dorsal horn of the spinal cord. However, these cells were encountered only very infrequently. They were also lightly stained and had few elongated processes. By E17, these two groups of TH-positive cells appeared considerably more mature. Both sets of cells not only stained more intensely, they also possessed more elaborate cellular processes. In addition, the population of cells ventral to the central canal also seemed more numerous (with commonly 5 or more cells observed per section) and were larger in size (8--.10 pm in diameter). The majority of the ventrally located cells had thick, well-stained processes that contacted the lumen of the central canal. Furthermore, a large number of these cells also possessed fine processes which originated from the side of the cells away from the canal. These processes were axon-likc in that they appeared varicose, yet they could only be followed over short distances. At EI 7, the number of cells found in the dorsal horn of the spinal cord increased dramatically over the few cells observed at E 15. Often one or more of these cells were observed per section at all levels of the cord. A striking feature of the anti-TH stained cells in the dorsal horn was that their size was often comparable to that of large motoneurons (both ranging from 20 to 25 pm in diameter). These large immunostained cells were frequently situated along the perimeter of the dorsal horn, especially near its boundary with the white matter of the lateral funiculus (Fig. 1A). Occasionally, stained cells were also found deeper within the dorsal horn near the preganglionic sympathetic cell column (nucleus of Terni) which is bilateral and dorsolateral to the central canal in thoracic and upper lumbar cord segments. At hatching (E21), the anti-TH-immunostained cells of the dorsal horn varied considerably in their morphology. While some appeared as spindle-shaped cells, others were pyrami-
255
dal or multipolar (Fig. I B), with several well-stained and variously branched processes. Few differences were noted in the appearance of immunostained cells ventral to the central canal between El7 and E21. C o m m o n to both ages, the processes that .
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Fig. 1. A: schematic diagram of a cross-section of the spinal cord at the junction of the lower thoracic and upper lumbar cord segment, l~[i, location of anti-T|l immunostained cells (dots) in the spinal cord of chick embryos at 17 days of incubation (El 7). The cells are situated along the perimeter of the dorsal horn adjacent to the lateral funiculus, near the nucleus of Terni (T), and ventral to the central canal (col. ( l h i s diagram represents a composite of approximately 6---8 sections (at 9/tin) to show the various positions of the dorsally located cells, not their relative density within a single section, llowever, for the cells shown ventral to the central canal, the number of cells illustrated is representative of their frequency that can be encountered in a single section.) Right. cytoarchitectonic organization of laminae within the lumbar cord of the chicken [13]. B: deeply-stained ceils in the dorsal horn at E21 appear pyramidal as well as multipolar, with easily visualized elaborate and branched processes. The cells shown in this tigure were from a section taken slightly oblique to the saginal plane of the cord due to unequal shrinkage of the face of the paralfin-embedded tissue. C': ventral to the central canal (*), several immunostained cells are seen. some of which retain their contact with the canal. However, as typified at E21 (the age shown m both (" and D), an increasing number of Tll-positive cells are also observed to have lost their contact with the central canal and are located more ventrally, further away from the canal. D: upon closer examination, the thick processes of the cells located immediately ventral to the canal have large club-like endings (arrow) within the lumen of the canal. Bars in B. (" = 25 itm: in D = I0 ltm.
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projected to the central canal ended in enlarged club-like swellings within the lume~ of the canal (Fig. I D). Howevcr, at E21 compared to El7, a greater number of the cells had lost contact with the lumen and were tbund more ventrally away from the canal, often considerably outside the ependymal cell layer (Fig. I C). The locations of TH-positive cells reported in this investigation correlate well wit h the description of CA neurons occurring within the spinal cords o1" other species. In particular, the immunostained cells vcntral to the central canal in the chick embryo appear analogous to cells demonstrated by CA histofluorcscence or by tmti-Tll immunocytochemistry in several lower vcrtebrate species [14, 15, 17, 24]. In contrast. the cells within the dorsal horn, described here, have not been observed in lower species, but have been found in almost identical locations by CA histofluorescence and anti-TH immunocytochemistry in the rat [3, 18]. Thus, it appears that the chicken is intermediate along the evolutionary line with respect to the occurrence and position o f these CA cells intrinsic to the spinal cord. Since catecholamines have been demonstrated histochemically within cells found ventral to the central canal in lower vertebrate species, as well as within cells of the dorsal horn of the spinal cord in higher vertebrate species, we examined whether the two populations of TH-positive cells in the chick spinal cord also possess CA. in these studies, we investigated mid-cervical and upper lumbar cord segments in chick
F i g 2. A: fluorescence micrograph of the upper lumbar spinal cord o f a hatchling (6 12 h old) demonstrating glyoxylic acid histofluorescence of catecholamines ICA) in the vicinity of the central canal (*) m a control animal without drug treatment. Note the large number of brightly fluorescent terminals surrounding the ependymal cells lining the central canal, but the absence of fluorescence within cells ventral to the canal where numerous "Ill-positive perikarya occur. B: at the same level of the cord in a hatchling treated with 300 mg/kg nialamide and 300 mg/kg L-DOPA (4 h prior to sacrifice), numerous CA-positive cells appear ventral to the canal in almosl every section On the same location as TH-immunostained cells observed at this age~. These CA-histofluore~ent cells possess thick-proces,~s (arrow) projecting towards anti contacting the lumen of the central canal. Bar 25 ilm.
25,'7
hatchlings (within 6 12 h of hatching). Utilizing the glyoxylic acid histofluorescencc technique (as described by Kimura et al. [I I]), no fluorescent cells could be visualized at either level of the cord in most control animals (Fig. 2A). (However, in a small number of cases, a few weakly fluorescent cells were found at both cord levels. These few CA-positive cells were located ventral to the central canal, but never in the dorsal horn.) In contrast, after pretreating chick hatchlings with a monoamine oxidase inhibitor (either nialamide or pargyline, at 150 300 mg..'kg, i.p.) in combination with the ('A synthesis precursor. L-I)OPA (l.-fl-3,4-dihydroxyphenylalaninc, at 150 300 m g k g , i.p.) for 4 h prior to sacrifice, far greater numbers of cells ventral to the canal were observed with brighter fluorescence (Fig. 2B) at both cord levels. After the same pharmacologic treatments, however, only a few cells in the dorsal horn were CApositive. These involved only those cells located ventrally near the nucleus of Terni. rather than within regions of the dorsal horn where the majority of TH-positivc cells were located. The conversion of the non-fluorescent compound. I.-DOPA, to a fluorescent ('A product, suggests that the TH-positive cells ventral to the central canal in the chick may also possess aromatic L-amino acid decarboxylase (AAI)C). the en~,yme that converts I - I ) O P A to the fluorescent product, dopamine. There are other reports of TH-positive neurons within the CNS that do not concomitantly contain detectable levels of catecholamines, nor A A D C [6, 10]. Furthermore, in the one case examined [I 0]. these TH-imrnunostained cells could not convert t.-DOPA to a fluorescent compound. Moreover, the locations of the cells mentioned in these previous studies (within the ventrolateral portions of the ventral horn of the spinal cord in the embryonic rat [6], and within the inferior colliculus of the postnatal rat pup [10]) do not have an ex.olutionary history of possessing catecholamines. In contrast, the FI lstained cells ventral to the central canal in the chick are comparable in location to dopamine-containing spinal cord cells in lower vertebrate species [14]. In a further cvohttionary context, the dorsally-situated TH-positive cells in the chick spinal cord are also of interest because they appear in higher vertebrate species [3. 18], rather than in lower vertebrate species. Overall, both populations of spinal cord TH-irnnlunoreactivc cells in the chick may represent a model in which to study factors rcgt.lating the expression of the ('A neurotransmitter phenotype in the developing CNS. The authors would like to thank Dr. William I)ail for his review of this article and to Susan H o m e r for her help in preparing the manuscript. This research was supported by NSF Grant BNS-8511079, N I H Grant NS-20039 and Minority' Biomedical Research Support Grant RR-08139.
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