Neuroscience Letters, 145 (1992) 71-74 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
71
NSL 08981
Co-localization of Met-enkephalin and somatostatin in the spinal cord of the rat A.J. T o d d a n d R.C. Spike Department of Anatomy, Universityof Glasgow, Glasgow ( UK) (Received 5 May 1992; Revised version received 30 June 1992; Accepted 30 June 1992)
Key words: Dorsal horn; Peptide; Irnmunohistochemistry A double-labelling immunofluorescence study of rat spinal dorsal horn was carried out with antisera to Met-enkephalin and somatostatin. Varicosities in laminae I and II were frequently immunoreactive with both antisera, and in addition some neuronal cell bodies in lamina II possessed both types of immunoreactivity. These findings suggest that enkephalin and somatostatin coexist in some axons within the rat superficial dorsal horn and that at least some of these axons are derived from local neurones.
Many neuropeptides are present in axons within the spinal cord, and these are particularly concentrated in laminae I and II [12]. Although many of these peptidecontaining axons are of primary afferent origin, and some may descend from cells in the brain, it is likely that many are derived from intrinsic neurones of the spinal dorsal horn. The locations of peptide-containing cells within the spinal cord were initially studied by immunohistochemistry, generally after the application of colchicine which prevents axoplasmic flow and therefore increases the concentration of peptide in the soma to detectable levels. However, colchicine may itself alter peptide synthesis, and this may result in detection of cells which would not normally synthesize a significant amount ofpeptide [8]. With more sensitive immunohistochemical methods, it is often possible to identify immunoreactive cell bodies in material from animals which have not been treated with colchicine. In addition, the technique of in situ hybridization histochemistry has allowed the demonstration of the cell bodies of neurones which synthesize the neuropeptide precursor proteins. Although many studies have demonstrated patterns of peptide coexistence in dorsal root ganglion cells, less is known about the coexistence of peptides in intrinsic spinal neurones. Several recent immunocytochemical studies have indicated that substance P coexists with Met-
Correspondence: A.J. Todd, Department of Anatomy, University of Glasgow, Glasgow G12 8QQ, UK.
enkephalin in cell bodies and axon terminals within the superficial dorsal horn [11, 14, 15], and that both peptides are even present within the same dense-cored vesicles [6]. Substance P- and dynorphin-like immunoreactivity (-LI) have also been found to coexist in varicosities i n rat superficial dorsal horn, and since the density of these varicosities was not altered by dorsal root section, the source of these fibres is likely to include intrinsic neurones in this area [18]. Met-enkephalin is produced from the precursor preproenkepahlin (PPE) and neurones containing Metenkephalin or PPE m R N A have been identified within the dorsal horn. Such cells are present in all dorsal horn laminae, but are most numerous in laminae I and II [4, 5, 13]. Enkephalin-immunoreactive fibres are also concentrated in laminae I and II, but are present in smaller numbers in deeper laminae of the dorsal horn, around the central canal and in the ventral horn [3]. Neurones which contain or synthesize somatostatin are also present in dorsal horn, but are more restricted to its superficial part, with only a few present in deeper layers [2, 5, 7]. Somatostatin-immunoreactive fibres are also mainly restricted to laminae I and II, although some fibres are present around the central canal and in the adjacent part of lamina VII [2]. Many of the somatostatin-immunoreactive axons within the dorsal horn are primary afferents, although it is thought that the majority are derived from intrinsic neurones [1]. Since there are similarities and differences in the distribution of cell bodies and fibres which contain the two neuropeptides, it is of
72 interest to know whether the peptides coexist within the dorsal horn. Three female Albino-Swiss rats (220-230 g) were anaesthetized with sodium pentobarbitone (60 mg, i.p.) and fixed by intracardiac perfusion with 4% formaldehyde in 0.1 M phosphate buffer. The L 4 segment of the spinal cord was rinsed and cryoprotected in 30% sucrose. Transverse sections 8-12/~m thick were cut with a cryostat, mounted onto slides and processed by a two-stage procedure for simultaneous detection of somatostatin and Met-enkephalin. The sections were incubated overnight in rat antiserum to somatostatin (Affiniti; diluted 1:150-250) and then in biotinylated anti-rat IgG (Sigma; diluted 1:100) and avidin-fluorescein (Vector; 20/lg/ml). Excess biotin or avidin binding was blocked by sequential treatment with avidin and biotin solutions and the sections were then incubated for 1 h in rabbit antiserum to Met-enkephalin (Penisula; 1:400), followed by biotinylated anti-rabbit lgG (Vector; diluted 1:200) and avidin-Texas red (Vector; 20/,tg/ml). All antibodies were diluted in 1% normal goat serum/0.3% Triton X-100. The fluorescence was viewed with a Nikon microscope fitted with filter sets selective for fluorescein or Texas red. Control sections were processed in the same way, except that one of the peptide antisera was omitted. In addition, some sections were incubated in primary antiserum which had been incubated for 1 h with either Metenkephalin or somatostatin (0.1/1tool peptide/25/11 diluted antiserum). The Met-enkepha]in antiserum is reported to show no cross-reactivity to Leu-enkephalin (manufacturer's specification) and is therefore likely to provide a reliable marker for cells which synthesize PPE. The patterns of immunostaining with the 2 antisera were similar to those reported previously with antisera to these peptides [1-3, 5]. In each case there was heavy staining of varicosities in laminae I and II (Fig. la,b). Small numbers of enkephalin-immunoreactive fibres were seen in the deeper laminae of the dorsal horn and in the ventral horn, and there were numerous fibres around the central canal. Somatostatin-immunoreactive fibres were less frequent in these areas, although some were seen around the central canal. With each antiserum a few immunoreactive cell bodies were present in lamina II (03/half section), but none were seen in other laminae. The immunostaining of cell bodies was restricted to perikaryal cytoplasm, and was often asymmetrical (Fig. lc-f). Within laminae I and II many varicosities were immunoreactive with both antisera, while others showed only somatostatin-LI or enkephalin-LI (Fig. lc-h). Double-labelled varicosities were present throughout the full thickness of laminae I and II. In addition, several cell bodies were immunoreactive with both antisera (Fig. lc f), while others were immunoreactive only with one an-
tiserum. In the 18 half sections in which cells were counted, 15 cell bodies which were immunoreactive with both antisera were observed. Omission of the primary antiserum or incubation with the corresponding peptide abolished the associated fluorescent staining. Incubation of the primary antiserum with the other peptide had no effect. The avidin-biotin-fluorescence method used in this study is sufficiently sensitive to allow staining of some peptide-containing cell bodies, without the need for application of colchicine. However, it is clear that only a small fraction of peptide-containing cells were detected by this method, and for this reason no attempt was made to determine the proportion of neuronal cell bodies which showed both types of immunoreactivity. Although both primary antibodies are detected by using biotinylated secondary antibodies and fluorochromes conjugated to avidin, there does not appear to be significant cross-reaction between the two stages of the reaction, since omission of either primary antiserum resulted in absence of the equivalent fluorescence and many profiles showed only one type of immunoreactivity. The present results strongly suggest that Met-enkephalin and somatostatin coexist in axons and cell bodies within the rat superficial dorsal horn. Some of the axons which contain both peptides may be derived from neurones in the medulla, since ceils in this region which project to the spinal cord may possess both enkephalin- and somatostatin-LI [10]. However, since both types of immunoreactivity were found to coexist in neurones in lamina II, it is likely that these cells are a major source of axons within the superficial dorsal horn which contain both enkephalin and somatostatin. Previous immunohistochemical studies have suggested that approximately 50% of enkephalin-containing neurones in laminae I and II also contain substance P [11, 14]. However, somatostatin- and substance P-LI are found in different populations of varicosities in rat superficial dorsal horn [18] and these two peptides therefore probably do not coexist in dorsal horn neurones. This suggests that at least two distinct populations of enkephalin-containing neurones are present in this region: those which contain enkephalin and substance P and those which contain enkephalin and somatostatin. In addition, we have recently demonstrated that many enkephalin-immunoreactive cells in superficial dorsal horn are GABA-immunoreactive [17], whereas somatostatin-immunoreactive cells in this area (like neurotensin-immunoreactive cells [16]) do not show GABA-LI (F. Proudlock, R. Spike and A. Todd, unpublished observations). Since substance P and GABA appear to be present in different axons in superficial dorsal horn [9], there may therefore be a third group of enkephalin-containing
73
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Fig. 1. Enkephalin-like (a, c, e and g) and somatostatin-like (b, d, fand h) immunoreactivity in rat dorsal horn. a and b show the same field with filters set for enkephalin- and somatostatin-like immunoreactivities, respectively. With both antisera, many immunoreactive varicosities present in laminae I and II and there are few in lamina Ilk c and d are higher magnification views to show part of the same field. A cell body which is immunoreactive with both antisera is visible (large arrows) and some of the varicosities also show both types of immunoreactivity (two are indicated with small arrows), while many other unmarked profiles are immunoreactive with only one of the antisera, e and f show another cell body (large arrows) and a varicosity (small arrows) which are immunoreactive with both antisera. Two other varicosities are identified with arrowheads: one of these is enkephalin-immunoreactive and the other is somatostatin-immunoreactive, g shows a cluster of enkephalin-immunoreactive varicosities. In h it can be seen that only one of these is also somatostatin-immunoreactive (arrow). One of those which does not show somatostatin-LI is indicated by an arrowhead in g. Bars = 50/.tm (b); 2 0 p m (d); 10pm (fand h).
74
neurones, which contain GABA, but not somatostatin or substance P. We are grateful to Mr. J. McGadey, Mr. D. Russell, Miss C. Morris and Miss M. Hughes for technical assistance. This work was supported by the Wellcome Trust. 1 Alvarez, F.J. and Priestley, J.V., Anatomy of somatostatin-immunoreactive fibres and cell bodies in the rat trigeminal subnucleus caudalis, Neuroscience, 38 (1990) 343-357. 2 Dalsgaard, C.-J., Hrkfelt, T., Johansson, O. and Elde, R., Somatostatin immunoreactive cell bodies in the dorsal horn and the parasympathetic intermediolateral nucleus of the rat spinal cord, Neurosci. Lett., 27 (1981) 335-339. 3 Gibson, S.J., Polak, J., Bloom, S.R. and Wall, ED., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. Comp. Neurol., 201 (1981) 65-80. 4 Hrkfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 5 Hunt, S.P., Kelly, J.S., Emson, EC., Kimmel, J.R., Miller, R.J. and Wu, J.-Y., An immunohistochemical study of neuronal populations containing neuropeptides or y-aminobutyrate within the superficial layers of the rat dorsal horn, Neuroscience, 6 ( 1981) 1883-1898. 6 Katoh, S., Hisano, S., Kawano, H., Kagotani, Y. and Daikoku, S., Light- and electron-microscopic evidence of costoring of immunoreactive enkephalins and substance P in dorsal horn neurons of rat, Cell Tissue Res., 253 (1988) 297-303. 7 Kiyama, H. and Emson, EC., Distribution of somatostatin mRNA in the rat nervous system as visualized by a novel non-radioactive in situ hybridization histochemistry procedure, Neuroscience, 38 (1990) 223-244. 8 Kiyama, H. and Emson, P.C., Colchicine-induced expression of proneurotensin mRNA in rat striatum and hypothalamus, Mol. Brain Res., 9 (1991) 353-358. 9 Merighi, A., Polak, J.M., Fumagatli, G, and Theodosis, D.T., UItrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques, J. Histochem. Cytochem., 37 (1989) 529-540.
10 Millhorn, D.E., Seroogy, K., Hrkfelt, T., Schmoed, L.C., Terenius, L., Buchan, A. and Brown, J.C., Neurons of the ventral medulla obolongata that contain both somatostatin and enkephalin immunoreactivies project to nucleus tractus solitarii and spinal cord, Brain Res., 424 (1987) 99-108. 11 Ribeiro-da-Silva, A., Pioro, E.P. and Cuello, A.C., Substance Pand enkephalin-like immunoreactivities are colocalized in certain neurons of the substantia gelatinosa of the rat spinal cord: an ultrastructural double-labeling study, J. Neurosci., 11 (1991) 1068-1080. 12 Ruda, M.A., Bennett, G.J. and Dubner, R., Neurochemistry and neural circuitry in the dorsal horn. In P.C. Emson, M.N. Rossor and M. Tohyama (Eds.), Progress in Brain Research, Vol. 66, Elsevier, Amsterdam, 1986, pp. 219-268. 13 Ruda, M.A., Iadarola, M.J., Cohen, L.V. and Young, W.S., In situ hybridization histochemistry and immunocytochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model Of inflammation and hyperalgesia, Proc. Natl. Acad. Sci. USA, 85 (1988) 622~526. 14 Senba, E., Yanaihara, C., Yanaihara, N. and Tohyama, M., Colocalization of substance P and Met-enkephalin-Arg6-Glyr-Leu8 in the intraspinal neurons of the rat, with special reference to the neurons of the substantia gelatinosa, Brain Res., 453 (1988) 110-116. 15 Tashiro, T., Takahashi, O., Satoda, T., Matsushima, R. and Mizuno, N., Immunohistochemical demonstration of coexistence of enkephalin- and substance P-like immunoreactivities in axonal components in the lumbar segments of cat spinal cord, Brain Res.. 424 (1987) 391-395. 16 Todd, A.J., Russell, G. and Spike, R.C., Immunocytochemical evidence that GABA and neurotensin exist in different neurons in laminae II and III of rat spinal dorsal horn, Neuroscience, 47 (1992) 685-691. 17 Todd, A.J., Spike, R.C., Russell, G. and Johnston, H.M., Immunohistochemical evidence that Met-enkephalin and GABA coexist in some neurones in rat dorsal horn, Brain Res., 584 (1992) 149-156. 18 Tuscherer, M.M. and Seybold, V.S., A quantitative study of the coexistence of peptides in varicosities within the superficial laminae of the dorsal horn of the rat spinal cord, J. Neurosci., 9 (1989) 195-205.
71
NSL 08981
Co-localization of Met-enkephalin and somatostatin in the spinal cord of the rat A.J. T o d d a n d R.C. Spike Department of Anatomy, Universityof Glasgow, Glasgow ( UK) (Received 5 May 1992; Revised version received 30 June 1992; Accepted 30 June 1992)
Key words: Dorsal horn; Peptide; Irnmunohistochemistry A double-labelling immunofluorescence study of rat spinal dorsal horn was carried out with antisera to Met-enkephalin and somatostatin. Varicosities in laminae I and II were frequently immunoreactive with both antisera, and in addition some neuronal cell bodies in lamina II possessed both types of immunoreactivity. These findings suggest that enkephalin and somatostatin coexist in some axons within the rat superficial dorsal horn and that at least some of these axons are derived from local neurones.
Many neuropeptides are present in axons within the spinal cord, and these are particularly concentrated in laminae I and II [12]. Although many of these peptidecontaining axons are of primary afferent origin, and some may descend from cells in the brain, it is likely that many are derived from intrinsic neurones of the spinal dorsal horn. The locations of peptide-containing cells within the spinal cord were initially studied by immunohistochemistry, generally after the application of colchicine which prevents axoplasmic flow and therefore increases the concentration of peptide in the soma to detectable levels. However, colchicine may itself alter peptide synthesis, and this may result in detection of cells which would not normally synthesize a significant amount ofpeptide [8]. With more sensitive immunohistochemical methods, it is often possible to identify immunoreactive cell bodies in material from animals which have not been treated with colchicine. In addition, the technique of in situ hybridization histochemistry has allowed the demonstration of the cell bodies of neurones which synthesize the neuropeptide precursor proteins. Although many studies have demonstrated patterns of peptide coexistence in dorsal root ganglion cells, less is known about the coexistence of peptides in intrinsic spinal neurones. Several recent immunocytochemical studies have indicated that substance P coexists with Met-
Correspondence: A.J. Todd, Department of Anatomy, University of Glasgow, Glasgow G12 8QQ, UK.
enkephalin in cell bodies and axon terminals within the superficial dorsal horn [11, 14, 15], and that both peptides are even present within the same dense-cored vesicles [6]. Substance P- and dynorphin-like immunoreactivity (-LI) have also been found to coexist in varicosities i n rat superficial dorsal horn, and since the density of these varicosities was not altered by dorsal root section, the source of these fibres is likely to include intrinsic neurones in this area [18]. Met-enkephalin is produced from the precursor preproenkepahlin (PPE) and neurones containing Metenkephalin or PPE m R N A have been identified within the dorsal horn. Such cells are present in all dorsal horn laminae, but are most numerous in laminae I and II [4, 5, 13]. Enkephalin-immunoreactive fibres are also concentrated in laminae I and II, but are present in smaller numbers in deeper laminae of the dorsal horn, around the central canal and in the ventral horn [3]. Neurones which contain or synthesize somatostatin are also present in dorsal horn, but are more restricted to its superficial part, with only a few present in deeper layers [2, 5, 7]. Somatostatin-immunoreactive fibres are also mainly restricted to laminae I and II, although some fibres are present around the central canal and in the adjacent part of lamina VII [2]. Many of the somatostatin-immunoreactive axons within the dorsal horn are primary afferents, although it is thought that the majority are derived from intrinsic neurones [1]. Since there are similarities and differences in the distribution of cell bodies and fibres which contain the two neuropeptides, it is of
72 interest to know whether the peptides coexist within the dorsal horn. Three female Albino-Swiss rats (220-230 g) were anaesthetized with sodium pentobarbitone (60 mg, i.p.) and fixed by intracardiac perfusion with 4% formaldehyde in 0.1 M phosphate buffer. The L 4 segment of the spinal cord was rinsed and cryoprotected in 30% sucrose. Transverse sections 8-12/~m thick were cut with a cryostat, mounted onto slides and processed by a two-stage procedure for simultaneous detection of somatostatin and Met-enkephalin. The sections were incubated overnight in rat antiserum to somatostatin (Affiniti; diluted 1:150-250) and then in biotinylated anti-rat IgG (Sigma; diluted 1:100) and avidin-fluorescein (Vector; 20/lg/ml). Excess biotin or avidin binding was blocked by sequential treatment with avidin and biotin solutions and the sections were then incubated for 1 h in rabbit antiserum to Met-enkephalin (Penisula; 1:400), followed by biotinylated anti-rabbit lgG (Vector; diluted 1:200) and avidin-Texas red (Vector; 20/,tg/ml). All antibodies were diluted in 1% normal goat serum/0.3% Triton X-100. The fluorescence was viewed with a Nikon microscope fitted with filter sets selective for fluorescein or Texas red. Control sections were processed in the same way, except that one of the peptide antisera was omitted. In addition, some sections were incubated in primary antiserum which had been incubated for 1 h with either Metenkephalin or somatostatin (0.1/1tool peptide/25/11 diluted antiserum). The Met-enkepha]in antiserum is reported to show no cross-reactivity to Leu-enkephalin (manufacturer's specification) and is therefore likely to provide a reliable marker for cells which synthesize PPE. The patterns of immunostaining with the 2 antisera were similar to those reported previously with antisera to these peptides [1-3, 5]. In each case there was heavy staining of varicosities in laminae I and II (Fig. la,b). Small numbers of enkephalin-immunoreactive fibres were seen in the deeper laminae of the dorsal horn and in the ventral horn, and there were numerous fibres around the central canal. Somatostatin-immunoreactive fibres were less frequent in these areas, although some were seen around the central canal. With each antiserum a few immunoreactive cell bodies were present in lamina II (03/half section), but none were seen in other laminae. The immunostaining of cell bodies was restricted to perikaryal cytoplasm, and was often asymmetrical (Fig. lc-f). Within laminae I and II many varicosities were immunoreactive with both antisera, while others showed only somatostatin-LI or enkephalin-LI (Fig. lc-h). Double-labelled varicosities were present throughout the full thickness of laminae I and II. In addition, several cell bodies were immunoreactive with both antisera (Fig. lc f), while others were immunoreactive only with one an-
tiserum. In the 18 half sections in which cells were counted, 15 cell bodies which were immunoreactive with both antisera were observed. Omission of the primary antiserum or incubation with the corresponding peptide abolished the associated fluorescent staining. Incubation of the primary antiserum with the other peptide had no effect. The avidin-biotin-fluorescence method used in this study is sufficiently sensitive to allow staining of some peptide-containing cell bodies, without the need for application of colchicine. However, it is clear that only a small fraction of peptide-containing cells were detected by this method, and for this reason no attempt was made to determine the proportion of neuronal cell bodies which showed both types of immunoreactivity. Although both primary antibodies are detected by using biotinylated secondary antibodies and fluorochromes conjugated to avidin, there does not appear to be significant cross-reaction between the two stages of the reaction, since omission of either primary antiserum resulted in absence of the equivalent fluorescence and many profiles showed only one type of immunoreactivity. The present results strongly suggest that Met-enkephalin and somatostatin coexist in axons and cell bodies within the rat superficial dorsal horn. Some of the axons which contain both peptides may be derived from neurones in the medulla, since ceils in this region which project to the spinal cord may possess both enkephalin- and somatostatin-LI [10]. However, since both types of immunoreactivity were found to coexist in neurones in lamina II, it is likely that these cells are a major source of axons within the superficial dorsal horn which contain both enkephalin and somatostatin. Previous immunohistochemical studies have suggested that approximately 50% of enkephalin-containing neurones in laminae I and II also contain substance P [11, 14]. However, somatostatin- and substance P-LI are found in different populations of varicosities in rat superficial dorsal horn [18] and these two peptides therefore probably do not coexist in dorsal horn neurones. This suggests that at least two distinct populations of enkephalin-containing neurones are present in this region: those which contain enkephalin and substance P and those which contain enkephalin and somatostatin. In addition, we have recently demonstrated that many enkephalin-immunoreactive cells in superficial dorsal horn are GABA-immunoreactive [17], whereas somatostatin-immunoreactive cells in this area (like neurotensin-immunoreactive cells [16]) do not show GABA-LI (F. Proudlock, R. Spike and A. Todd, unpublished observations). Since substance P and GABA appear to be present in different axons in superficial dorsal horn [9], there may therefore be a third group of enkephalin-containing
73
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•
¢
Fig. 1. Enkephalin-like (a, c, e and g) and somatostatin-like (b, d, fand h) immunoreactivity in rat dorsal horn. a and b show the same field with filters set for enkephalin- and somatostatin-like immunoreactivities, respectively. With both antisera, many immunoreactive varicosities present in laminae I and II and there are few in lamina Ilk c and d are higher magnification views to show part of the same field. A cell body which is immunoreactive with both antisera is visible (large arrows) and some of the varicosities also show both types of immunoreactivity (two are indicated with small arrows), while many other unmarked profiles are immunoreactive with only one of the antisera, e and f show another cell body (large arrows) and a varicosity (small arrows) which are immunoreactive with both antisera. Two other varicosities are identified with arrowheads: one of these is enkephalin-immunoreactive and the other is somatostatin-immunoreactive, g shows a cluster of enkephalin-immunoreactive varicosities. In h it can be seen that only one of these is also somatostatin-immunoreactive (arrow). One of those which does not show somatostatin-LI is indicated by an arrowhead in g. Bars = 50/.tm (b); 2 0 p m (d); 10pm (fand h).
74
neurones, which contain GABA, but not somatostatin or substance P. We are grateful to Mr. J. McGadey, Mr. D. Russell, Miss C. Morris and Miss M. Hughes for technical assistance. This work was supported by the Wellcome Trust. 1 Alvarez, F.J. and Priestley, J.V., Anatomy of somatostatin-immunoreactive fibres and cell bodies in the rat trigeminal subnucleus caudalis, Neuroscience, 38 (1990) 343-357. 2 Dalsgaard, C.-J., Hrkfelt, T., Johansson, O. and Elde, R., Somatostatin immunoreactive cell bodies in the dorsal horn and the parasympathetic intermediolateral nucleus of the rat spinal cord, Neurosci. Lett., 27 (1981) 335-339. 3 Gibson, S.J., Polak, J., Bloom, S.R. and Wall, ED., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. Comp. Neurol., 201 (1981) 65-80. 4 Hrkfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 5 Hunt, S.P., Kelly, J.S., Emson, EC., Kimmel, J.R., Miller, R.J. and Wu, J.-Y., An immunohistochemical study of neuronal populations containing neuropeptides or y-aminobutyrate within the superficial layers of the rat dorsal horn, Neuroscience, 6 ( 1981) 1883-1898. 6 Katoh, S., Hisano, S., Kawano, H., Kagotani, Y. and Daikoku, S., Light- and electron-microscopic evidence of costoring of immunoreactive enkephalins and substance P in dorsal horn neurons of rat, Cell Tissue Res., 253 (1988) 297-303. 7 Kiyama, H. and Emson, EC., Distribution of somatostatin mRNA in the rat nervous system as visualized by a novel non-radioactive in situ hybridization histochemistry procedure, Neuroscience, 38 (1990) 223-244. 8 Kiyama, H. and Emson, P.C., Colchicine-induced expression of proneurotensin mRNA in rat striatum and hypothalamus, Mol. Brain Res., 9 (1991) 353-358. 9 Merighi, A., Polak, J.M., Fumagatli, G, and Theodosis, D.T., UItrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques, J. Histochem. Cytochem., 37 (1989) 529-540.
10 Millhorn, D.E., Seroogy, K., Hrkfelt, T., Schmoed, L.C., Terenius, L., Buchan, A. and Brown, J.C., Neurons of the ventral medulla obolongata that contain both somatostatin and enkephalin immunoreactivies project to nucleus tractus solitarii and spinal cord, Brain Res., 424 (1987) 99-108. 11 Ribeiro-da-Silva, A., Pioro, E.P. and Cuello, A.C., Substance Pand enkephalin-like immunoreactivities are colocalized in certain neurons of the substantia gelatinosa of the rat spinal cord: an ultrastructural double-labeling study, J. Neurosci., 11 (1991) 1068-1080. 12 Ruda, M.A., Bennett, G.J. and Dubner, R., Neurochemistry and neural circuitry in the dorsal horn. In P.C. Emson, M.N. Rossor and M. Tohyama (Eds.), Progress in Brain Research, Vol. 66, Elsevier, Amsterdam, 1986, pp. 219-268. 13 Ruda, M.A., Iadarola, M.J., Cohen, L.V. and Young, W.S., In situ hybridization histochemistry and immunocytochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model Of inflammation and hyperalgesia, Proc. Natl. Acad. Sci. USA, 85 (1988) 622~526. 14 Senba, E., Yanaihara, C., Yanaihara, N. and Tohyama, M., Colocalization of substance P and Met-enkephalin-Arg6-Glyr-Leu8 in the intraspinal neurons of the rat, with special reference to the neurons of the substantia gelatinosa, Brain Res., 453 (1988) 110-116. 15 Tashiro, T., Takahashi, O., Satoda, T., Matsushima, R. and Mizuno, N., Immunohistochemical demonstration of coexistence of enkephalin- and substance P-like immunoreactivities in axonal components in the lumbar segments of cat spinal cord, Brain Res.. 424 (1987) 391-395. 16 Todd, A.J., Russell, G. and Spike, R.C., Immunocytochemical evidence that GABA and neurotensin exist in different neurons in laminae II and III of rat spinal dorsal horn, Neuroscience, 47 (1992) 685-691. 17 Todd, A.J., Spike, R.C., Russell, G. and Johnston, H.M., Immunohistochemical evidence that Met-enkephalin and GABA coexist in some neurones in rat dorsal horn, Brain Res., 584 (1992) 149-156. 18 Tuscherer, M.M. and Seybold, V.S., A quantitative study of the coexistence of peptides in varicosities within the superficial laminae of the dorsal horn of the rat spinal cord, J. Neurosci., 9 (1989) 195-205.
