Cell and Tissue Research
Cell Tissue Res. 200, 83-90 (1979)
9 by Springer-Verlag 1979
Somatostatinergic Nerves in the Cervical Spinal Cord of the Monkey C. Burnweit* and W.G. Forssmann** Department of Anatomy, University of Heidelberg, Heidelberg, Federal Republic of Germany
Summary. Somatostatinergic nerves in the spinal cord of the m o n k e y were investigated utilizing immunohistochemistry with various antibodies against synthetic somatostatin. In contrast to earlier investigations, it is shown that somatostatinergic nerve endings occur in most of the areas of the grey matter of the spinal cord. The somatostatinergic axons are, however, characteristically distributed in three main regions: (1) Densely-packed endings are seen in lamina II of the substantia gelatinosa, forming a crescent-shaped pattern in the columna dorsalis. Somatostatin immunoreactivity is also seen in lamina I and in the Lissauer tract. (2) A fine network of fibers is observed around the central canal; the endings are concentrated on special cell bodies. Some single perikarya are also stained in this region. (3) A loose network of single fibers is found ending on perikarya of the columna lateralis or ventralis. The perikarya of the nerve axons, with the exception of those terminating in the columna dorsalis, have as yet not been identified. In order to better understand the somatostatinergic system of the spinal cord, these newlydetected somatostatinergic nerves must be studied and their exact pathways analyzed. Key words: Spinal cord -
Tupaia
-
Somatostatin - Immunohistochemistry.
G r o w t h hormone release-inhibiting factor or somatostatin, initially described as a hypothalamic tetradecapeptide inhibiting the secretion of growth hormones from the pituitary (Brazeau et al., 1973), has been detected in m a n y extrahypothalamic tissues in the last few years. This peptide is suggested to act both as a hormone and as a neurotransmitter and is present in three main systems: (1) central nervous system, (2) peripheral somatosensible and autonomic nervous system and (3) endocrine glands such as the pancreas, gut and thyroid (see Luft et al., 1978). The evidence that somatostatin is widely distributed in the spinal cord has been proven Prof. Dr. W.G. Forssmann, Anatomisches Institut III, der Universit/~t, Im Neuenheimer Feld 307, D-6900 Heidelberg 1, Federal Republic of Germany * Summer student from Harvard Medical School, Boston (USA) ** We are indebted to Miss Karin Biehl and Mrs. Hedwig Traub for technical assistance and to Mrs. Rosa Botz and Miss Anita Miehle for preparing the manuscript S e n d oJJprint requests to:
0302-766X/79/0200/0083/$01.60
84
c. Burnweit and W.G. Forssmann
b y b o t h r a d i o i m m u n o a s s a y a n d i m m u n o h i s t o c h e m i s t r y . It was s h o w n t h a t r a d i o i m m u n o a s s a y a b l e s o m a t o s t a t i n is m a i n l y c o n c e n t r a t e d in the c o l u m n a d o r s a l i s t h r o u g h o u t all s e g m e n t s o f the spinal c o r d . I n a d d i t i o n , i m m u n o h i s t o c h e m i s t r y s h o w e d t h a t s o m a t o s t a t i n - s p e c i f i c i m m u n o r e a c t i v i t y is r e s t r i c t e d to the s u b s t a n t i a g e l a t i n o s a o r l a m i n a e I a n d II in the c o l u m n a d o r s a l i s ( H 6 k f e l t et al., 1975, 1976). T h i s p o s i t i v e r e a c t i o n p r o b a b l y r e p r e s e n t s n e r v e e n d i n g s o f p r i m a r y s e n s o r y n e u r o n s , t h e cell b o d i e s o f w h i c h lie in the spinal g a n g l i a a n d also stain f o r s o m a t o s t a t i n by f l u o r e s c e n t i m m u n o h i s t o c h e m i s t r y t e c h n i q u e s . F u r t h e r m o r e , it is m e n t i o n e d t h a t s o m a t o s t a t i n - i m m u n o r e a c t i v e fibers a r e seen in the L i s s a u e r t r a c t a n d in a d j a c e n t a r e a s o f the f u n i c u l u s lateralis ( H 6 k f e l t et al., 1976). U s i n g v a r i o u s i m m u n o h i s t o c h e m i c a l m e t h o d s , we h a v e a n a l y z e d t h e e x a c t d i s t r i b u t i o n o f s o m a t o s t a t i n e r g i c s t r u c t u r e s in the spinal c o r d o f t h e m o n k e y (Tupaia belangeri) a n d h a v e o b t a i n e d d i f f e r e n t results, e.g., n e r v e fibers in o t h e r a r e a s o f the s p i n a l c o r d in a d d i t i o n to t h o s e a l r e a d y d e s c r i b e d .
M a t e r i a l s and M e t h o d s
Three adult tree shrews (Tupaia belanger0 were investigated. The animals were anesthetized with Nembutal and a perfusion fixation was carried out through the cannulated aorta abdominalis (for technical details see Forssmann et al., 1977). Rinsing solution containing procaine-hydrochloride was applied for 45 s, the fixation perfusion was run for 4-5min, and a 10min rinsing with PBS-buffer solution followed. The entire brain and spinal cord was dissected and carefully freed from the bony parts of the skull and vertebrae. The tissue was dehydrated in ethanol and xylol and embedded in paraffin. In the case of one animal, the entire spinal cord from C3 to C1 and the brain were serially sectioned at a thickness of 10 lam. The sections were deparaffinized with xylol and incubated with serum from rabbits immunized against synthetic somatostatin. The antisera were previously tested and also shown to react specifically with gut and pancreatic somatostatin (D) cells and with the nerve axons of the hypothalamic infundibulum. An incubation with either immunofluorescence (IF) according to Coons et al. (1955) or peroxidase-antiperoxidase (PAP) according to the Sternberger (1974) labelling system followed. For the evaluations in this study, only PAP-stained sections were used because the resolution of staining is much better than with IF. At intervals of 50 sections, two sections were stained and the nerve fibers completely mapped in schematic transectional drawings. A Zeiss Axiomat was used for both position charting and photography.
Control Reactions The immunoreactivity technique was tested by several experiments, all showing that the reaction was specific for somatostatin. Antisera of rabbits immunized against other neuropeptides and GEPhormones showed no reactivity (e.g. gastrin antiserum) or a different pattern of reactivity (e.g. neurotensin antiserum). Positive staining was obtained by dilutions of the somatostatin antiserum up to 1 : 5000. The absorption with 1 ~tg synthetic somatostatin per 1 Ixl of somatostatin antiserum diluted 1 : 5000 resulted in complete supression of the staining for somatostatin. Comparable amounts of other GEP-hormones and neuropeptides added to the somatostatin antiserum did not affect the somatostatin immunoreactivity.
Results
I n v e s t i g a t e d s e g m e n t s o f the u p p e r c e r v i c a l spinal c o r d are s h o w n in Fig. 11. A s seen in p r a c t i c a l l y all s e c t i o n s o f this p a r t o f t h e c e n t r a l n e r v o u s system, the s o m a t o s t a t i n
Figs. 1 and 2. C o l u m n a dorsalis showing intensely reactive, crescent-like immunoreactive nerve endings indicating the somatostatinergic system in lamina I and II. x 80 and x 920 Fig. 3. Higher magnification of somatostatin immunoreactive nerve fibers in the columna ventralis of segment C3. Several varicosities of somatostatin immunoreactive nerves are observed (arrows). • 410 Figs. 4 and 5. Somatostatin immunoreactive nerve fibers travelling from the columna dorsalis to the substantia intermedia centralis close to the fasciculus cuneatus and reaching the area around the central
86
C. Burnweit and W.G. Forssmann
immunoreactive axons are widely distributed in the grey matter and occasionally traverse the white columns (Fig. 1-10). However, the axons exhibiting a positive reaction are densely packed in the columna dorsalis of the spinal cord and this area is readily seen with the naked eye or in low power micrographs (Fig. 1 and 2). In the columna dorsalis, somatostatinergic axons are localized in three distinct regions: (1) The highest intensity is seen in lamina II or the substantia gelatinosa. The immunoreactive structures appear as round dots of various sizes between 0.05 to 0.4 gm. These positive structures are usually displayed as strands of beads or single beads (Figs. 1 and 2). (2) Smaller similar structures are also seen in lamina I and in the adjacent tractus dorsolateralis (Lissauer). (3) Positive reacting smaller strands of beads are seen more frequently laterally along lamina I and lamina II, next to the funiculus lateralis. These stained structures are often intermingled with the white matter of the columna dorsalis. Furthermore, distinct fibers of lamina X around the central canal regularly exhibit a positive reaction with somatostatin staining (Figs. 9 and 10). They form a loose network, the fibers of which are frequently beaded and may traverse the median line through the commisura dorsalis or ventralis. It could be clearly demonstrated that the density of this network is scarce in C3 and constantly increases to C 1 and the spinal cord medulla junction (compare Figs. 9 and 10). Neurons with intensely stained perikarya have also been detected in the substantia intermedia centralis of the columna lateralis (lamina X and the medial part of lamina VII). These perikarya are mostly 10-20 gm large and exhibit stained axon-like extensions (Figs. 6 and 7). The number of these neurons steadily increases from C 3 to C 1 : in C 3, one of four sections contains a positive neuron; in C 2, about one neuron per section appears; and in C1, two to three neurons are stained per section. Further localizations of the somatostatinergic varicosities are found in the medial part of lamina II-VI, directly adjacent to the fasciculus cuneatus (Figs. 4 and 5) in the columna lateralis dorsal to the substantia intermedia centralis. In addition, positive fibers are seen in lamina IX (Fig. 3), sometimes forming distinct basket-like structures around the large neurons of the columna ventralis. Some thin beaded fibers are also observed in lamina VII, e.g. extending through the entire substantia intermedia centralis et lateralis (Fig. 6), occasionally reaching the neighbouring white matter of the funiculus lateralis. Fig. 6. Higher magnification of the substantia intermedia lateralis showing numerous varicosities which indicate the presence of somatostatinergic nerves in this area. x 580 Figs. 7 and 8. Somatostatin immunoreactive perikarya seen in C 1 indicating somatostatinergic nerve cell bodies (large arrows) and slender axons from these nerve ceils (small arrows). The nerve cell bodies are found in the substantia intermedia centralis, lateral to the canalis centralis. Several other varicosities in the substantia intermedia with somatostatin immunoreactivity are also seen (double arrows). • 410 Figs. 9 and 10. Somatostatin immunoreactive nerve fibers in the substantia gelatinosa centralis around the canalis centralis seen in the cervical spinal cord C 1 (Fig. 9) and C 3 (Fig. 10) close to the commissura grisea anterior and posterior (arrows). One somatostatin nerve reaching this area from the columna dorsalis is also seen (double arrow), x 410
Somatostatinergic Nerves in Spinal Cord
87
88
C. Burnweit and W.G. F o r s s m a n n
~,--". ~'~ ;t '. ~:~
S-462
/ \
\\
\\\ ~
I
I C1
~i.. ;,....~i! s:- 9..) :.
/" --
."
C2 C3
//
/
/
/
/
/
l Fig. 11. Distribution of somatostatin immunoreactive structures in CI, C2 and C3. Further explanations see text
SomatostatinergicNervesin Spinal Cord
89
The complete results of this study are summarized in Fig. 11 showing the extent of the somatostatinergic systems of the cervical spinal cord in Tupaia. Discussion
The somatostatin immunoreactive structures mapped in serial sections of the cervical spinal segments have been found to be much more extensive than previously described (see H6kfelt et al., 1975, 1976; Luft et al., 1978). Using similar methods of immunohistochemical staining for somatostatin, other authors have found nerve fibers (H6kfelt et al., 1974, 1975; Dub~ et al., 1975; King et al., 1975; Pelletier et al., 1975; S~thl6 et al., 1975) and pefikarya (Dubois and Kolodziejzyk, 1975; Elde and Parsons, 1975; Alpert et al., 1976; Kfisch, 1979) in many areas of the brain. It is still a controversial matter whether perikarya are stained under normal conditions or only if axonal transport is inhibited by colchicine. It has been suggested that the inhibition ofaxonal transport results in an accumulation of these peptides at the sites of synthesis and thereby make the perikarya stainable. In our investigation, however, antisera used in conjunction with IF and PAP seemed adequate for staining perikarya as well as nerve varicosifies. Using the same methods, we could stain nerve cell bodies and nerve fibers in the telencephalon and brain stem (Forssmann and Burnweit, 1979; unpublished observations) of species such as rat, mouse, and monkey. In this study, we describe neurons of the spinal cord which are for the first time shown to stain positive for somatostatin. These perikarya are located in lamina X, in the medial areas of lamina VII and sometimes in the nucleus dorsomedialis (Clarke). The fibers of such perikarya seem to form part of a heretofore uncharted pathway of somatostatinergic fibers. These fibers around the canalis centralis, however, seem to stem from other neurons as well. The staining of certain perikarya is ostensively displayed by the staining of a basket-like array of dense varicosities ending on a perikaryon, as mentioned by Petrusz et al. (1978). Although it appears that the staining of cytoplasm within perikarya can be distinguished from that of positive varicosities around nonstained perikarya, we need more accurate methods to establish that somatostatinergic neurons are present in the cervical spinal cord. To date, other authors have described somatostatinergic nerves in only the columna dorsalis of the spinal cord (see H6kfelt et al., 1975, 1976). The additional staining found in this study seems to be due to: (1) the technique and species used in our work and (2) the segment of the spinal cord investigated. The above-mentioned authors do not indicate whether they investigated C 1 to C3 in serial sections. The number of somatostatinergic structures considerably increases towards the spinal cord-medulla junction. Initially we investigated some cross-sections of spinal cord of various animals without carefully determining the segments (Forssmann, 1978). In these investigations we did not find somatostatinergic structures other than those described by H6kfelt et al. (1975, 1976). This work, however, shows that careful mapping throughout the whole spinal cord is necessary to define the somatostatinergic system in this part of the central nervous system. Preliminary investigations of the medulla oblongata showed that somatostatinergic structures are more extensively distributed (Forssmann and Burnweit, 1979) than was previously believed. The functional implications of these findings may be essential, but we still
90
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need more information on the exact topographical pathways of somatostatinergic nerves before a discussion of this matter is meaningful.
References Alpert, L.C., Brawer, J.R., Patel, Y.C., Reichlin, S.: Somatostatinergic neurons in anterior hypothalamus: immunohistochemical localization. Endocrinology 98, 255-258 (1976) Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R.: Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hromone. Science 179, 77-79 (1973) Coons, A.H., Leduc, E.H., Conolly, J.M.: Studies on antibody production. I. Methods for histochemical demonstration of specific antibody and its application to a study of hyperimmune rabbit. J. Exp. Med. 102, 49-60 (1955) Dubr, D., Leclerc, R., Pelletier, G., Arimura, A., Schally, A.V.: Immunohistochemical detection of growth hormone-release inhibiting hormone (somatostatin) in the guinea-pig brain. Cell Tissue Res. 161, 385-392 (1975) Dubois, M.P., Kolodziejczyk, E.: Centres hypothalamiques du rat srcr&ant la somatostatine: rrpartition des prricaryons en 2 syst+mes magno et parvocellulaires (&ude immunocytologique). C. R. Acad. Sci. (D), Paris, 281, 1737-1740 (1975) Elde, R.P., Parsons, J.A.: Immunocytochemical localization of somatostatin in cell bodies of the rat hypothalamus. Am. J. Anat. 144, 541-548 (1975) Forssmann, W.G.: A new somatostatinergic system in the mammalian spinal cord. Neuroscience Letters 10, 293-297 (1978) Forssmann, W.G., Burnweit, C.: New somatostatinergic systems in the medulla oblongata of the monkey. Cell Tissue Res. (in preparation) Forssmann, W.G., Ito, S., Weihe, E., Aoki, A., Dym, M., Fawcett, D.W.: An improved perfusion fixation method for the testis. Anat. Rec. 188, 307-314 (1977) H6kfelt, T., Efendir, S., Johansson, O., Luft, R., Arimura, A.: Immunohistochemical localization of somatostatin (growth hormone release-inhibiting factor) in the guinea pig brain. Brain Res. 80, 165169 (1974) H6kfelt, T., Efendic, S., Hellerstr6m, C., Johansson, O., Luft, R., Arimura, A.: Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the A 1-cell of the pancreatic islets and to the hypothalamus. Acta Endocrinol. [Suppl.] (Kbh.) 200, 541 (1975a) H6kfelt, T., Elde, R., Johansson, O., Luft, R., Arimura, A.: Immunohistochemical evidence for the presence of somatostatin, a powerful inhibitory peptide, in some primary sensory neurons. Neuroscience Letters 1,231-235 (1975b) H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G., Arimura, A.: Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat. Neuroscience 1, 131-136 (1976) King, J.C., Arimura, A., Gerall, A.G., Fishback, J.B., Elking, K.E.: Growth hormone-release inhibiting hormone (GH-RIH) pathway of the rat hypothalamus revealed by the unlabeled antibody peroxidase-antiperoxidase method. Cell Tissue Res. 160, 423430 (1975) Krisch, B.: Hypothalamic and extrahypothalamic distribution of somatostatin-immunoreactive elements in the rat brain. Cell Tissue Res. 195, 499-513 (1978) Luft, R., Efendir, S., Hrkfelt, T.: Somatostatin- both hormone and neurotransmitter ? Diabetologia 14, 1-13 (1978) Pelletier, G., Leclerc, R., Dubr, D., Labrie, F., Puviani, R., Arimura, A., Schally, A.V.: Localization of growth hormone-release-inhibiting hormone (somatostatin) in the rat brain. Am. J. Anat. 142, 397401 (1975) Petrusz, P., Sar, M., Grossman, G.H., Kizer, J.S.: Synaptic terminals with somatostatin-like immunoreactivity in the rat brain. Brain Res. 137, 181-187 (1977) Srtfil6, G., Vigh, S., Schally, A.V., Arimura, A., Flerkr, B.: GH-RIH-containing neural elements in the rat hypothalamus. Brain Res. 90, 352-356 (1975) Sternberger, L.A.: Immunocytochemistry. Englewood Cliffs, New Jersey: Prentice-Hall 1974 Accepted April 24, 1979
Cell Tissue Res. 200, 83-90 (1979)
9 by Springer-Verlag 1979
Somatostatinergic Nerves in the Cervical Spinal Cord of the Monkey C. Burnweit* and W.G. Forssmann** Department of Anatomy, University of Heidelberg, Heidelberg, Federal Republic of Germany
Summary. Somatostatinergic nerves in the spinal cord of the m o n k e y were investigated utilizing immunohistochemistry with various antibodies against synthetic somatostatin. In contrast to earlier investigations, it is shown that somatostatinergic nerve endings occur in most of the areas of the grey matter of the spinal cord. The somatostatinergic axons are, however, characteristically distributed in three main regions: (1) Densely-packed endings are seen in lamina II of the substantia gelatinosa, forming a crescent-shaped pattern in the columna dorsalis. Somatostatin immunoreactivity is also seen in lamina I and in the Lissauer tract. (2) A fine network of fibers is observed around the central canal; the endings are concentrated on special cell bodies. Some single perikarya are also stained in this region. (3) A loose network of single fibers is found ending on perikarya of the columna lateralis or ventralis. The perikarya of the nerve axons, with the exception of those terminating in the columna dorsalis, have as yet not been identified. In order to better understand the somatostatinergic system of the spinal cord, these newlydetected somatostatinergic nerves must be studied and their exact pathways analyzed. Key words: Spinal cord -
Tupaia
-
Somatostatin - Immunohistochemistry.
G r o w t h hormone release-inhibiting factor or somatostatin, initially described as a hypothalamic tetradecapeptide inhibiting the secretion of growth hormones from the pituitary (Brazeau et al., 1973), has been detected in m a n y extrahypothalamic tissues in the last few years. This peptide is suggested to act both as a hormone and as a neurotransmitter and is present in three main systems: (1) central nervous system, (2) peripheral somatosensible and autonomic nervous system and (3) endocrine glands such as the pancreas, gut and thyroid (see Luft et al., 1978). The evidence that somatostatin is widely distributed in the spinal cord has been proven Prof. Dr. W.G. Forssmann, Anatomisches Institut III, der Universit/~t, Im Neuenheimer Feld 307, D-6900 Heidelberg 1, Federal Republic of Germany * Summer student from Harvard Medical School, Boston (USA) ** We are indebted to Miss Karin Biehl and Mrs. Hedwig Traub for technical assistance and to Mrs. Rosa Botz and Miss Anita Miehle for preparing the manuscript S e n d oJJprint requests to:
0302-766X/79/0200/0083/$01.60
84
c. Burnweit and W.G. Forssmann
b y b o t h r a d i o i m m u n o a s s a y a n d i m m u n o h i s t o c h e m i s t r y . It was s h o w n t h a t r a d i o i m m u n o a s s a y a b l e s o m a t o s t a t i n is m a i n l y c o n c e n t r a t e d in the c o l u m n a d o r s a l i s t h r o u g h o u t all s e g m e n t s o f the spinal c o r d . I n a d d i t i o n , i m m u n o h i s t o c h e m i s t r y s h o w e d t h a t s o m a t o s t a t i n - s p e c i f i c i m m u n o r e a c t i v i t y is r e s t r i c t e d to the s u b s t a n t i a g e l a t i n o s a o r l a m i n a e I a n d II in the c o l u m n a d o r s a l i s ( H 6 k f e l t et al., 1975, 1976). T h i s p o s i t i v e r e a c t i o n p r o b a b l y r e p r e s e n t s n e r v e e n d i n g s o f p r i m a r y s e n s o r y n e u r o n s , t h e cell b o d i e s o f w h i c h lie in the spinal g a n g l i a a n d also stain f o r s o m a t o s t a t i n by f l u o r e s c e n t i m m u n o h i s t o c h e m i s t r y t e c h n i q u e s . F u r t h e r m o r e , it is m e n t i o n e d t h a t s o m a t o s t a t i n - i m m u n o r e a c t i v e fibers a r e seen in the L i s s a u e r t r a c t a n d in a d j a c e n t a r e a s o f the f u n i c u l u s lateralis ( H 6 k f e l t et al., 1976). U s i n g v a r i o u s i m m u n o h i s t o c h e m i c a l m e t h o d s , we h a v e a n a l y z e d t h e e x a c t d i s t r i b u t i o n o f s o m a t o s t a t i n e r g i c s t r u c t u r e s in the spinal c o r d o f t h e m o n k e y (Tupaia belangeri) a n d h a v e o b t a i n e d d i f f e r e n t results, e.g., n e r v e fibers in o t h e r a r e a s o f the s p i n a l c o r d in a d d i t i o n to t h o s e a l r e a d y d e s c r i b e d .
M a t e r i a l s and M e t h o d s
Three adult tree shrews (Tupaia belanger0 were investigated. The animals were anesthetized with Nembutal and a perfusion fixation was carried out through the cannulated aorta abdominalis (for technical details see Forssmann et al., 1977). Rinsing solution containing procaine-hydrochloride was applied for 45 s, the fixation perfusion was run for 4-5min, and a 10min rinsing with PBS-buffer solution followed. The entire brain and spinal cord was dissected and carefully freed from the bony parts of the skull and vertebrae. The tissue was dehydrated in ethanol and xylol and embedded in paraffin. In the case of one animal, the entire spinal cord from C3 to C1 and the brain were serially sectioned at a thickness of 10 lam. The sections were deparaffinized with xylol and incubated with serum from rabbits immunized against synthetic somatostatin. The antisera were previously tested and also shown to react specifically with gut and pancreatic somatostatin (D) cells and with the nerve axons of the hypothalamic infundibulum. An incubation with either immunofluorescence (IF) according to Coons et al. (1955) or peroxidase-antiperoxidase (PAP) according to the Sternberger (1974) labelling system followed. For the evaluations in this study, only PAP-stained sections were used because the resolution of staining is much better than with IF. At intervals of 50 sections, two sections were stained and the nerve fibers completely mapped in schematic transectional drawings. A Zeiss Axiomat was used for both position charting and photography.
Control Reactions The immunoreactivity technique was tested by several experiments, all showing that the reaction was specific for somatostatin. Antisera of rabbits immunized against other neuropeptides and GEPhormones showed no reactivity (e.g. gastrin antiserum) or a different pattern of reactivity (e.g. neurotensin antiserum). Positive staining was obtained by dilutions of the somatostatin antiserum up to 1 : 5000. The absorption with 1 ~tg synthetic somatostatin per 1 Ixl of somatostatin antiserum diluted 1 : 5000 resulted in complete supression of the staining for somatostatin. Comparable amounts of other GEP-hormones and neuropeptides added to the somatostatin antiserum did not affect the somatostatin immunoreactivity.
Results
I n v e s t i g a t e d s e g m e n t s o f the u p p e r c e r v i c a l spinal c o r d are s h o w n in Fig. 11. A s seen in p r a c t i c a l l y all s e c t i o n s o f this p a r t o f t h e c e n t r a l n e r v o u s system, the s o m a t o s t a t i n
Figs. 1 and 2. C o l u m n a dorsalis showing intensely reactive, crescent-like immunoreactive nerve endings indicating the somatostatinergic system in lamina I and II. x 80 and x 920 Fig. 3. Higher magnification of somatostatin immunoreactive nerve fibers in the columna ventralis of segment C3. Several varicosities of somatostatin immunoreactive nerves are observed (arrows). • 410 Figs. 4 and 5. Somatostatin immunoreactive nerve fibers travelling from the columna dorsalis to the substantia intermedia centralis close to the fasciculus cuneatus and reaching the area around the central
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immunoreactive axons are widely distributed in the grey matter and occasionally traverse the white columns (Fig. 1-10). However, the axons exhibiting a positive reaction are densely packed in the columna dorsalis of the spinal cord and this area is readily seen with the naked eye or in low power micrographs (Fig. 1 and 2). In the columna dorsalis, somatostatinergic axons are localized in three distinct regions: (1) The highest intensity is seen in lamina II or the substantia gelatinosa. The immunoreactive structures appear as round dots of various sizes between 0.05 to 0.4 gm. These positive structures are usually displayed as strands of beads or single beads (Figs. 1 and 2). (2) Smaller similar structures are also seen in lamina I and in the adjacent tractus dorsolateralis (Lissauer). (3) Positive reacting smaller strands of beads are seen more frequently laterally along lamina I and lamina II, next to the funiculus lateralis. These stained structures are often intermingled with the white matter of the columna dorsalis. Furthermore, distinct fibers of lamina X around the central canal regularly exhibit a positive reaction with somatostatin staining (Figs. 9 and 10). They form a loose network, the fibers of which are frequently beaded and may traverse the median line through the commisura dorsalis or ventralis. It could be clearly demonstrated that the density of this network is scarce in C3 and constantly increases to C 1 and the spinal cord medulla junction (compare Figs. 9 and 10). Neurons with intensely stained perikarya have also been detected in the substantia intermedia centralis of the columna lateralis (lamina X and the medial part of lamina VII). These perikarya are mostly 10-20 gm large and exhibit stained axon-like extensions (Figs. 6 and 7). The number of these neurons steadily increases from C 3 to C 1 : in C 3, one of four sections contains a positive neuron; in C 2, about one neuron per section appears; and in C1, two to three neurons are stained per section. Further localizations of the somatostatinergic varicosities are found in the medial part of lamina II-VI, directly adjacent to the fasciculus cuneatus (Figs. 4 and 5) in the columna lateralis dorsal to the substantia intermedia centralis. In addition, positive fibers are seen in lamina IX (Fig. 3), sometimes forming distinct basket-like structures around the large neurons of the columna ventralis. Some thin beaded fibers are also observed in lamina VII, e.g. extending through the entire substantia intermedia centralis et lateralis (Fig. 6), occasionally reaching the neighbouring white matter of the funiculus lateralis. Fig. 6. Higher magnification of the substantia intermedia lateralis showing numerous varicosities which indicate the presence of somatostatinergic nerves in this area. x 580 Figs. 7 and 8. Somatostatin immunoreactive perikarya seen in C 1 indicating somatostatinergic nerve cell bodies (large arrows) and slender axons from these nerve ceils (small arrows). The nerve cell bodies are found in the substantia intermedia centralis, lateral to the canalis centralis. Several other varicosities in the substantia intermedia with somatostatin immunoreactivity are also seen (double arrows). • 410 Figs. 9 and 10. Somatostatin immunoreactive nerve fibers in the substantia gelatinosa centralis around the canalis centralis seen in the cervical spinal cord C 1 (Fig. 9) and C 3 (Fig. 10) close to the commissura grisea anterior and posterior (arrows). One somatostatin nerve reaching this area from the columna dorsalis is also seen (double arrow), x 410
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SomatostatinergicNervesin Spinal Cord
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The complete results of this study are summarized in Fig. 11 showing the extent of the somatostatinergic systems of the cervical spinal cord in Tupaia. Discussion
The somatostatin immunoreactive structures mapped in serial sections of the cervical spinal segments have been found to be much more extensive than previously described (see H6kfelt et al., 1975, 1976; Luft et al., 1978). Using similar methods of immunohistochemical staining for somatostatin, other authors have found nerve fibers (H6kfelt et al., 1974, 1975; Dub~ et al., 1975; King et al., 1975; Pelletier et al., 1975; S~thl6 et al., 1975) and pefikarya (Dubois and Kolodziejzyk, 1975; Elde and Parsons, 1975; Alpert et al., 1976; Kfisch, 1979) in many areas of the brain. It is still a controversial matter whether perikarya are stained under normal conditions or only if axonal transport is inhibited by colchicine. It has been suggested that the inhibition ofaxonal transport results in an accumulation of these peptides at the sites of synthesis and thereby make the perikarya stainable. In our investigation, however, antisera used in conjunction with IF and PAP seemed adequate for staining perikarya as well as nerve varicosifies. Using the same methods, we could stain nerve cell bodies and nerve fibers in the telencephalon and brain stem (Forssmann and Burnweit, 1979; unpublished observations) of species such as rat, mouse, and monkey. In this study, we describe neurons of the spinal cord which are for the first time shown to stain positive for somatostatin. These perikarya are located in lamina X, in the medial areas of lamina VII and sometimes in the nucleus dorsomedialis (Clarke). The fibers of such perikarya seem to form part of a heretofore uncharted pathway of somatostatinergic fibers. These fibers around the canalis centralis, however, seem to stem from other neurons as well. The staining of certain perikarya is ostensively displayed by the staining of a basket-like array of dense varicosities ending on a perikaryon, as mentioned by Petrusz et al. (1978). Although it appears that the staining of cytoplasm within perikarya can be distinguished from that of positive varicosities around nonstained perikarya, we need more accurate methods to establish that somatostatinergic neurons are present in the cervical spinal cord. To date, other authors have described somatostatinergic nerves in only the columna dorsalis of the spinal cord (see H6kfelt et al., 1975, 1976). The additional staining found in this study seems to be due to: (1) the technique and species used in our work and (2) the segment of the spinal cord investigated. The above-mentioned authors do not indicate whether they investigated C 1 to C3 in serial sections. The number of somatostatinergic structures considerably increases towards the spinal cord-medulla junction. Initially we investigated some cross-sections of spinal cord of various animals without carefully determining the segments (Forssmann, 1978). In these investigations we did not find somatostatinergic structures other than those described by H6kfelt et al. (1975, 1976). This work, however, shows that careful mapping throughout the whole spinal cord is necessary to define the somatostatinergic system in this part of the central nervous system. Preliminary investigations of the medulla oblongata showed that somatostatinergic structures are more extensively distributed (Forssmann and Burnweit, 1979) than was previously believed. The functional implications of these findings may be essential, but we still
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need more information on the exact topographical pathways of somatostatinergic nerves before a discussion of this matter is meaningful.
References Alpert, L.C., Brawer, J.R., Patel, Y.C., Reichlin, S.: Somatostatinergic neurons in anterior hypothalamus: immunohistochemical localization. Endocrinology 98, 255-258 (1976) Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R.: Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hromone. Science 179, 77-79 (1973) Coons, A.H., Leduc, E.H., Conolly, J.M.: Studies on antibody production. I. Methods for histochemical demonstration of specific antibody and its application to a study of hyperimmune rabbit. J. Exp. Med. 102, 49-60 (1955) Dubr, D., Leclerc, R., Pelletier, G., Arimura, A., Schally, A.V.: Immunohistochemical detection of growth hormone-release inhibiting hormone (somatostatin) in the guinea-pig brain. Cell Tissue Res. 161, 385-392 (1975) Dubois, M.P., Kolodziejczyk, E.: Centres hypothalamiques du rat srcr&ant la somatostatine: rrpartition des prricaryons en 2 syst+mes magno et parvocellulaires (&ude immunocytologique). C. R. Acad. Sci. (D), Paris, 281, 1737-1740 (1975) Elde, R.P., Parsons, J.A.: Immunocytochemical localization of somatostatin in cell bodies of the rat hypothalamus. Am. J. Anat. 144, 541-548 (1975) Forssmann, W.G.: A new somatostatinergic system in the mammalian spinal cord. Neuroscience Letters 10, 293-297 (1978) Forssmann, W.G., Burnweit, C.: New somatostatinergic systems in the medulla oblongata of the monkey. Cell Tissue Res. (in preparation) Forssmann, W.G., Ito, S., Weihe, E., Aoki, A., Dym, M., Fawcett, D.W.: An improved perfusion fixation method for the testis. Anat. Rec. 188, 307-314 (1977) H6kfelt, T., Efendir, S., Johansson, O., Luft, R., Arimura, A.: Immunohistochemical localization of somatostatin (growth hormone release-inhibiting factor) in the guinea pig brain. Brain Res. 80, 165169 (1974) H6kfelt, T., Efendic, S., Hellerstr6m, C., Johansson, O., Luft, R., Arimura, A.: Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the A 1-cell of the pancreatic islets and to the hypothalamus. Acta Endocrinol. [Suppl.] (Kbh.) 200, 541 (1975a) H6kfelt, T., Elde, R., Johansson, O., Luft, R., Arimura, A.: Immunohistochemical evidence for the presence of somatostatin, a powerful inhibitory peptide, in some primary sensory neurons. Neuroscience Letters 1,231-235 (1975b) H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G., Arimura, A.: Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat. Neuroscience 1, 131-136 (1976) King, J.C., Arimura, A., Gerall, A.G., Fishback, J.B., Elking, K.E.: Growth hormone-release inhibiting hormone (GH-RIH) pathway of the rat hypothalamus revealed by the unlabeled antibody peroxidase-antiperoxidase method. Cell Tissue Res. 160, 423430 (1975) Krisch, B.: Hypothalamic and extrahypothalamic distribution of somatostatin-immunoreactive elements in the rat brain. Cell Tissue Res. 195, 499-513 (1978) Luft, R., Efendir, S., Hrkfelt, T.: Somatostatin- both hormone and neurotransmitter ? Diabetologia 14, 1-13 (1978) Pelletier, G., Leclerc, R., Dubr, D., Labrie, F., Puviani, R., Arimura, A., Schally, A.V.: Localization of growth hormone-release-inhibiting hormone (somatostatin) in the rat brain. Am. J. Anat. 142, 397401 (1975) Petrusz, P., Sar, M., Grossman, G.H., Kizer, J.S.: Synaptic terminals with somatostatin-like immunoreactivity in the rat brain. Brain Res. 137, 181-187 (1977) Srtfil6, G., Vigh, S., Schally, A.V., Arimura, A., Flerkr, B.: GH-RIH-containing neural elements in the rat hypothalamus. Brain Res. 90, 352-356 (1975) Sternberger, L.A.: Immunocytochemistry. Englewood Cliffs, New Jersey: Prentice-Hall 1974 Accepted April 24, 1979
