Brain Research, 575 (1992) 151-154 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92l$05.00
151
BRES 25096
Evidence that fine primary afferent axons innervate a wider territory in the superficial dorsal horn following peripheral axotomy Adrian A. Cameron, Carolyn M. Pover, William D. Willis and Richard E. Coggeshall Department of Anatomy and Neurosciences, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Accepted 10 December 1991) Key words: Dorsal horn; Fine primary afferent fiber; Peripheral axotomy
Peripheral axotomy initiates changes in central primary afferent receiving areas of the dorsal horn of the spinal cord. Most of the presently known changes are degenerative in nature and consist of such things as cell and axon death or declines in peptides or enzymes. Other changes are regenerative in nature and because most of these occur in the superficial dorsal horn, which is where fine primary afferents end, we wished to ask whether peripheral axotomy results in a change in the distribution in these fine afferents. Using recently available markers for fine primary afferent axons and small dorsal root ganglion cells, we demonstrate that peripheral axotomy results in a considerable increase in the immunolabeled area for these compounds. Our interpretation is that there may be an extension of fine primary afferent fibers into lamina III and possibly lamina IV following peripheral axotomy. If further work bears out this conclusion, this would provide a possible explanation for the chronic pain states that sometimes follow peripheral nerve damage. Peripheral axotomy in adult mammals initiates a series of changes in primary afferent receiving areas of the spinal cord. Most of the changes that have been described are degenerative or regressive in nature. For example, following peripheral nerve section some dorsal root ganglion cells die and their central axons undergo Wallerian degeneration 1'6. A n o t h e r type of degenerative change, transganglionic degenerative atrophy, has also been reported 7. There are also marked depletions of various peptides and enzymes in the cord following peripheral axotomy 2'5'14. 'Regenerative' changes are less frequently discussed, but there are central increases in the immunostaining for such peptides as dynorphin, galanin and vasoactive intestinal polypeptide 3'8'12, and the appearance of a growth associated protein (GAP-43) in spinal laminae I and II 4' 15. Although these phenomena may not have a c o m m o n mechanism, the localization of GAP-43 and the various peptides and enzymes in the superficial dorsal horn suggests that fine sensory fibers are likely to be involved. Accordingly we wish to ask whether the distribution of fine primary afferents changes after peripheral axotomy. Several markers are now available that are reported to be specific for fine afferents and their terminals. A m o n g these are the lectin, soybean agglutinin (SBA) 1°, and an antibody to another lectin derived from rat lung (RL29) H. The present study reports that the spinal labeling by these compounds increases significantly in ex-
tent following peripheral axotomy. These results are interpreted as further evidence that some of the central changes that follow peripheral axotomy are regenerative in nature. Experiments were performed on 10 male SpragueDawley rats. The rats were anesthetized with sodium pentobarbital (50 mg/kg), and one sciatic nerve was exposed in mid-thigh. The nerve was ligated at two levels 3 mm apart and the isolated 3 m m segment was removed. The wound was sutured and the animals recovered uneventfully. After 14 days, the animals were reanesthetized with pentobarbital (70 mg/kg) and perfused through the aorta with 500 ml of normal saline containing 500 I U of heparin. This was followed by 500 ml of 4% paraformaldehyde in 0.1 M, p H 7.4, phosphate buffer (PB) in 4 animals and with 4% paraformaldehyde and 4% glutaraldehyde in PB in 6 animals. The L 4 and L 5 spinal segments were removed, a ventral notch was placed in the cord on the side opposite to the sciatic nerve transection, and the cord was postfixed overnight in the same fixative. The spinal cord was sectioned transversely at 30/~m using a vibratome. To test for SBA, the sections were incubated in a solution of 10/xg/ml of biotinylated S B A in a buffered salt solution 1°. After rinsing in the buffered salt solution, the sections were incubated in an avidin-biotin-horseradish peroxidase complex (Vector) in PB. After rinsing, the sections were incubated in a 0.05% solution of diaminobenzidine in PB
Correspondence: R.E. Coggeshall, Marine Biomedical Institute, 200 University Boulevard, Galveston, TX 77550, U.S.A.
152
A
': T :
Fig. 1. Light micrographs of immunostained dorsal horns. A: SBA staining. B: RL29 immunostaining. Note that there is intensive staining in the superficial dorsal horn in both A and B, but that there is a considerable increase in density and areal extent of immunostaining in the medial part of the immunostained areas on the left as compared to the right hand side of each side of each cord section. The left hand side of each cord section is the side of the sciatic nerve transection. Bar = 100/~m.
containing 0.015% H202. To test for RL29, the sections were incubated in rabbit antibody diluted 1:1000 in phosphate buffered saline (PBS) containing 0.3% triton-X and 2% normal goat serum. A f t e r rinsing in PBS, the sections were incubated in biotinylated goat anti-rabbit antibody diluted 1:200, rinsed in PBS and then placed in the a v i d i n - b i o t i n - h o r s e r a d i s h peroxidase complex for 1 h. A f t e r a final rinse in PBS, the sections were incubated in 0.05% diaminobenzidine in 0.1 M PB containing 0.015% H202. The sections were then m o u n t e d onto subbed slides and examined in a c o m p o u n d light microscope. To measure the size of the labeled areas, the o p e r a t e d and u n o p e r a t e d sides of each spinal cord section were digitized with an A m e r s h a m computer-assisted image analysis system ( A m e r s h a m Corporation, Research Analysis System, Version 1.00, 1988) interfaced with a
Nikon microscope via a video camera. The larger labeled area was used to d e t e r m i n e an optical density 'window' whose area is m e a s u r e d in pixels. E q u a l optical densities were then established and measured on the other side of each cord section. The area of label in each of the above regions was measured, and the percent difference between o p e r a t e d and u n o p e r a t e d sides determined. Mean differences from 3 sections for each antibody from each animal were then calculated. In addition, the labeling for RL29 was obviously m o r e intense in central parts of the i m m u n o l a b e l e d area. For these animals, a second smaller optical density window was set to measure only this m o r e intensely labeled area. A final point here is that there was an obvious increase in density of label, at least in laminae III and IV, because there was significant staining in these areas on the o p e r a t e d side but only background staining on the u n o p e r a t e d side.
153 TABLE I Percent increase in area o f label on operated as compared to unoperated sides
A table presenting the average amount of areal enlargement of the immunolabeled dorsal horn on the operated as compared to the unoperated side of the dorsal horn of the rat spinal cord. The measurements for SBA and RL29 are for the whole immunolabeled dorsal horn. By contrast the measurements for RL29, intensely labeled area, are only for the medial half of each dorsal horn. Note that the differences are much more dramatic for the latter measurements, n = 10. SBA
RL29
RL29 Intensely labeled area
35% (P < 0.001)
77% (P < 0.001)
216% (P < 0.001)
For each animal, labeling for S B A and RL29 on the unoperated side was found in the tract of Lissauer and in laminae I and II of the superficial dorsal horn (Fig. 1). O n the experimental side, the labeling was found in these same areas. In addition, however, S B A reactivity extended into much of lamina I I I and some of lamina IV medially (Fig. 1A). For RL29, the labeling extended primarily into lamina III and only minimally into the dorsal part of lamina IV medially (Fig. 1B). The pixel measurements, expressed as the % increase of operated to control sides of the immunostained areas, are presented in Table I. It should be stated that the differences are somewhat dependent on fixation in that the material with low glutaraldehyde shows greater differences, but the differences are still significant with either fixation. The overall measurements for S B A and RL29 were made on the entire immunolabeled area of the dorsal horn, but the differences are most pronounced in the medial 2/3s of the dorsal horn. This is illustrated by the striking side to side differences for the intensely labeled area immunostained for RL29, which was measured only in the medial half of the dorsal horn. In addition, as mentioned above, there was a considerable increase in density of label in the deeper laminae on the operated side in that there was only background label on the normal side. Our current results indicate that transection of the sciatic nerve in the rat leads to an increased area of im-
1 Arvidsson, J.,Ygge, J. and Grant, G., Cell loss in lumbar dorsal root ganglia and transganglionic degeneration after sciatic nerve resection in rat, Brain Res., 373 (1986) 15-21. 2 B~rbut, D., Polak, J.M. and Wall, ED., Substance P in spinal cord dorsal horn decreases following peripheral nerve injury, Brain Res., 205 (1981) 289-298. 3 Cho, H.J. and Basbaum, A.I., Ultrastructural analysis of dynorphin-B immunoreactive cells and terminals in the superficial dorsal horn of the rat, J. Comp. Neurol., 281 (1989) 193-205.
munoreactivity for two lectins, S B A and RL29, in those segments of the spinal cord where afferents from the sciatic nerve are known to end 5'13. In particular, the S B A labeling extends into lamina III and IV and the RL29 label essentially into lamina III medially, which is not the case on the unoperated side. In addition, particularly for the RL29 reaction, there is also an increase in intensity of staining on the operated side. Previous studies show that S B A and RL29 are found predominantly in small dorsal root ganglion ( D R G ) cells and unmyelinated afferent axons 9-11. Because of this and also because the increase in area is in the region where sciatic nerve axons distribute 5, we believe the simplest explanation of our data is that fine sciatic primary afferent fibers extend over a wider territory following peripheral axotomy. We presume this is an active 'regenerative' response to the vacated synaptic sites caused by the death of some D R G cells that follows peripheral axotomy 1'6. It should be emphasized that other interpretations are possible, however, and one possible source of confusion is that our RL29 material shows some label in the dorsal columns where large primary afferent fibers are located. Thus it will be necessary to provide further evidence that it is f i n e primary afferent fibers that are extending over wider areas by using, for example, transganglionic markers for fine primary afferent fibers coupled with electron microscopic examination of the tissue. At present, however, our interpretation is that there is an expansion of fine primary afferent fibers into lamina III and to a lesser extent into lamina IV following peripheral axotomy. Studies designed to determine the functional consequences of this phenomenon will be of considerable interest. In particular, we wish to ask whether cells that do not normally respond to noxious input in laminae III and possibly IV now do so. If this could be demonstrated, it would provide a possible explanation for the chronic pain states that sometimes follow peripheral axotomy.
This work is supported by NIH Grants NS 10161, NS 11255 and NS 09743 and the Bristol-Myers Squibb Corp. We wish to thank Drs. S. Barondes and H. Leflter for donating the antibody to RL29.
4 Coggeshall, R.E., Reynolds, M.L. and Woolf, C.J., Distribution of GAP-43 in the central processes of axotomized primary afferents in the adult spinal cord; presence of growth cone-like structures, Neurosci. Lett., 131 (1991) 37-41. 5 Devor, M. and Claman, D., Mapping and plasticity of acid phosphatase afferents in rat dorsal horn, Brain Res., 190 (1980) 17-28. 6 Klein, C.M., Guillamondegui, O., Krenek, C.D., LaForte, R.A. and Coggeshall, R.E., Do neuropeptides in the dorsal
154
7
8
9
10
11
horn change if the dorsal root ganglion cell death that normally accompanies peripheral nerve transection is prevented?, Brain Res., 552 (1991) 273-383. Knyihar-Csillik, E., Rakic, P. and Csillik, B., Transganglionic degenerative atrophy in the substantia gelatinosa of the spinal cord after peripheral nerve transection in rhesus monkeys, Cell Tissue Res., 247 (1987) 599-604. McGregor, G.P., Gibson, S.J., Sabate, I.M., Blank, M.A., Christofides, N.D., Wall, P.D., Polak, J.M. and Bloom, S.R., Effect of peripheral nerve section and nerve crush on spinal cord neuropeptides in the rat; increased VIP and PHI in the dorsal horn, Neuroscience, 13 (1984) 207-216. Plenderleith, M.B., Cameron, A.A., Key, B. and Snow, P.J., Soybean agglutinin binds to a subpopulation of primary sensory neurons in the cat, Neurosci. Lett., 86 (1988) 257-262. Plenderleith, M.B., Cameron, A.A., Key, B. and Snow, P.J., The plant lectin soybean agglutinin binds to the soma, axon and terminals of a subpopulation of small-diameter primary sensory neurons in the rat and cat, Neuroscience, 31 (1989) 683-695. Regan, L.J., Dodd, J., Barondes, S.H. and Jessell, T.M., Selective expression of lactose-binding lectins and lactoseries gly-
12
13
14
15
coconjugates in subsets of rat sensory neurons, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 2248-2252. Shehab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy, Exp. Brain Res., 62 (1986) 422-430. Swett, J.E. and Woolf, C.J., The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord, J. Comp. Neurol., 231 (1985) 66-77.11. Villar, M.J., Cortes, R., Theodorsson, E., Wiesenfeld-Hallin, Z., Schalling, M., Fahrenkrug, J., Emson, P.C. and H/)kfelt, T., Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin, Neuroscience, 33 (1989) 587-604. Woolf, C.J., Reynolds, M.L., Molander, C., O'Brien, C., Lindsay, R.M. and Benowitz, L.I., The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury, Neuroscience, 34 (1990) 465-478.
151
BRES 25096
Evidence that fine primary afferent axons innervate a wider territory in the superficial dorsal horn following peripheral axotomy Adrian A. Cameron, Carolyn M. Pover, William D. Willis and Richard E. Coggeshall Department of Anatomy and Neurosciences, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Accepted 10 December 1991) Key words: Dorsal horn; Fine primary afferent fiber; Peripheral axotomy
Peripheral axotomy initiates changes in central primary afferent receiving areas of the dorsal horn of the spinal cord. Most of the presently known changes are degenerative in nature and consist of such things as cell and axon death or declines in peptides or enzymes. Other changes are regenerative in nature and because most of these occur in the superficial dorsal horn, which is where fine primary afferents end, we wished to ask whether peripheral axotomy results in a change in the distribution in these fine afferents. Using recently available markers for fine primary afferent axons and small dorsal root ganglion cells, we demonstrate that peripheral axotomy results in a considerable increase in the immunolabeled area for these compounds. Our interpretation is that there may be an extension of fine primary afferent fibers into lamina III and possibly lamina IV following peripheral axotomy. If further work bears out this conclusion, this would provide a possible explanation for the chronic pain states that sometimes follow peripheral nerve damage. Peripheral axotomy in adult mammals initiates a series of changes in primary afferent receiving areas of the spinal cord. Most of the changes that have been described are degenerative or regressive in nature. For example, following peripheral nerve section some dorsal root ganglion cells die and their central axons undergo Wallerian degeneration 1'6. A n o t h e r type of degenerative change, transganglionic degenerative atrophy, has also been reported 7. There are also marked depletions of various peptides and enzymes in the cord following peripheral axotomy 2'5'14. 'Regenerative' changes are less frequently discussed, but there are central increases in the immunostaining for such peptides as dynorphin, galanin and vasoactive intestinal polypeptide 3'8'12, and the appearance of a growth associated protein (GAP-43) in spinal laminae I and II 4' 15. Although these phenomena may not have a c o m m o n mechanism, the localization of GAP-43 and the various peptides and enzymes in the superficial dorsal horn suggests that fine sensory fibers are likely to be involved. Accordingly we wish to ask whether the distribution of fine primary afferents changes after peripheral axotomy. Several markers are now available that are reported to be specific for fine afferents and their terminals. A m o n g these are the lectin, soybean agglutinin (SBA) 1°, and an antibody to another lectin derived from rat lung (RL29) H. The present study reports that the spinal labeling by these compounds increases significantly in ex-
tent following peripheral axotomy. These results are interpreted as further evidence that some of the central changes that follow peripheral axotomy are regenerative in nature. Experiments were performed on 10 male SpragueDawley rats. The rats were anesthetized with sodium pentobarbital (50 mg/kg), and one sciatic nerve was exposed in mid-thigh. The nerve was ligated at two levels 3 mm apart and the isolated 3 m m segment was removed. The wound was sutured and the animals recovered uneventfully. After 14 days, the animals were reanesthetized with pentobarbital (70 mg/kg) and perfused through the aorta with 500 ml of normal saline containing 500 I U of heparin. This was followed by 500 ml of 4% paraformaldehyde in 0.1 M, p H 7.4, phosphate buffer (PB) in 4 animals and with 4% paraformaldehyde and 4% glutaraldehyde in PB in 6 animals. The L 4 and L 5 spinal segments were removed, a ventral notch was placed in the cord on the side opposite to the sciatic nerve transection, and the cord was postfixed overnight in the same fixative. The spinal cord was sectioned transversely at 30/~m using a vibratome. To test for SBA, the sections were incubated in a solution of 10/xg/ml of biotinylated S B A in a buffered salt solution 1°. After rinsing in the buffered salt solution, the sections were incubated in an avidin-biotin-horseradish peroxidase complex (Vector) in PB. After rinsing, the sections were incubated in a 0.05% solution of diaminobenzidine in PB
Correspondence: R.E. Coggeshall, Marine Biomedical Institute, 200 University Boulevard, Galveston, TX 77550, U.S.A.
152
A
': T :
Fig. 1. Light micrographs of immunostained dorsal horns. A: SBA staining. B: RL29 immunostaining. Note that there is intensive staining in the superficial dorsal horn in both A and B, but that there is a considerable increase in density and areal extent of immunostaining in the medial part of the immunostained areas on the left as compared to the right hand side of each side of each cord section. The left hand side of each cord section is the side of the sciatic nerve transection. Bar = 100/~m.
containing 0.015% H202. To test for RL29, the sections were incubated in rabbit antibody diluted 1:1000 in phosphate buffered saline (PBS) containing 0.3% triton-X and 2% normal goat serum. A f t e r rinsing in PBS, the sections were incubated in biotinylated goat anti-rabbit antibody diluted 1:200, rinsed in PBS and then placed in the a v i d i n - b i o t i n - h o r s e r a d i s h peroxidase complex for 1 h. A f t e r a final rinse in PBS, the sections were incubated in 0.05% diaminobenzidine in 0.1 M PB containing 0.015% H202. The sections were then m o u n t e d onto subbed slides and examined in a c o m p o u n d light microscope. To measure the size of the labeled areas, the o p e r a t e d and u n o p e r a t e d sides of each spinal cord section were digitized with an A m e r s h a m computer-assisted image analysis system ( A m e r s h a m Corporation, Research Analysis System, Version 1.00, 1988) interfaced with a
Nikon microscope via a video camera. The larger labeled area was used to d e t e r m i n e an optical density 'window' whose area is m e a s u r e d in pixels. E q u a l optical densities were then established and measured on the other side of each cord section. The area of label in each of the above regions was measured, and the percent difference between o p e r a t e d and u n o p e r a t e d sides determined. Mean differences from 3 sections for each antibody from each animal were then calculated. In addition, the labeling for RL29 was obviously m o r e intense in central parts of the i m m u n o l a b e l e d area. For these animals, a second smaller optical density window was set to measure only this m o r e intensely labeled area. A final point here is that there was an obvious increase in density of label, at least in laminae III and IV, because there was significant staining in these areas on the o p e r a t e d side but only background staining on the u n o p e r a t e d side.
153 TABLE I Percent increase in area o f label on operated as compared to unoperated sides
A table presenting the average amount of areal enlargement of the immunolabeled dorsal horn on the operated as compared to the unoperated side of the dorsal horn of the rat spinal cord. The measurements for SBA and RL29 are for the whole immunolabeled dorsal horn. By contrast the measurements for RL29, intensely labeled area, are only for the medial half of each dorsal horn. Note that the differences are much more dramatic for the latter measurements, n = 10. SBA
RL29
RL29 Intensely labeled area
35% (P < 0.001)
77% (P < 0.001)
216% (P < 0.001)
For each animal, labeling for S B A and RL29 on the unoperated side was found in the tract of Lissauer and in laminae I and II of the superficial dorsal horn (Fig. 1). O n the experimental side, the labeling was found in these same areas. In addition, however, S B A reactivity extended into much of lamina I I I and some of lamina IV medially (Fig. 1A). For RL29, the labeling extended primarily into lamina III and only minimally into the dorsal part of lamina IV medially (Fig. 1B). The pixel measurements, expressed as the % increase of operated to control sides of the immunostained areas, are presented in Table I. It should be stated that the differences are somewhat dependent on fixation in that the material with low glutaraldehyde shows greater differences, but the differences are still significant with either fixation. The overall measurements for S B A and RL29 were made on the entire immunolabeled area of the dorsal horn, but the differences are most pronounced in the medial 2/3s of the dorsal horn. This is illustrated by the striking side to side differences for the intensely labeled area immunostained for RL29, which was measured only in the medial half of the dorsal horn. In addition, as mentioned above, there was a considerable increase in density of label in the deeper laminae on the operated side in that there was only background label on the normal side. Our current results indicate that transection of the sciatic nerve in the rat leads to an increased area of im-
1 Arvidsson, J.,Ygge, J. and Grant, G., Cell loss in lumbar dorsal root ganglia and transganglionic degeneration after sciatic nerve resection in rat, Brain Res., 373 (1986) 15-21. 2 B~rbut, D., Polak, J.M. and Wall, ED., Substance P in spinal cord dorsal horn decreases following peripheral nerve injury, Brain Res., 205 (1981) 289-298. 3 Cho, H.J. and Basbaum, A.I., Ultrastructural analysis of dynorphin-B immunoreactive cells and terminals in the superficial dorsal horn of the rat, J. Comp. Neurol., 281 (1989) 193-205.
munoreactivity for two lectins, S B A and RL29, in those segments of the spinal cord where afferents from the sciatic nerve are known to end 5'13. In particular, the S B A labeling extends into lamina III and IV and the RL29 label essentially into lamina III medially, which is not the case on the unoperated side. In addition, particularly for the RL29 reaction, there is also an increase in intensity of staining on the operated side. Previous studies show that S B A and RL29 are found predominantly in small dorsal root ganglion ( D R G ) cells and unmyelinated afferent axons 9-11. Because of this and also because the increase in area is in the region where sciatic nerve axons distribute 5, we believe the simplest explanation of our data is that fine sciatic primary afferent fibers extend over a wider territory following peripheral axotomy. We presume this is an active 'regenerative' response to the vacated synaptic sites caused by the death of some D R G cells that follows peripheral axotomy 1'6. It should be emphasized that other interpretations are possible, however, and one possible source of confusion is that our RL29 material shows some label in the dorsal columns where large primary afferent fibers are located. Thus it will be necessary to provide further evidence that it is f i n e primary afferent fibers that are extending over wider areas by using, for example, transganglionic markers for fine primary afferent fibers coupled with electron microscopic examination of the tissue. At present, however, our interpretation is that there is an expansion of fine primary afferent fibers into lamina III and to a lesser extent into lamina IV following peripheral axotomy. Studies designed to determine the functional consequences of this phenomenon will be of considerable interest. In particular, we wish to ask whether cells that do not normally respond to noxious input in laminae III and possibly IV now do so. If this could be demonstrated, it would provide a possible explanation for the chronic pain states that sometimes follow peripheral axotomy.
This work is supported by NIH Grants NS 10161, NS 11255 and NS 09743 and the Bristol-Myers Squibb Corp. We wish to thank Drs. S. Barondes and H. Leflter for donating the antibody to RL29.
4 Coggeshall, R.E., Reynolds, M.L. and Woolf, C.J., Distribution of GAP-43 in the central processes of axotomized primary afferents in the adult spinal cord; presence of growth cone-like structures, Neurosci. Lett., 131 (1991) 37-41. 5 Devor, M. and Claman, D., Mapping and plasticity of acid phosphatase afferents in rat dorsal horn, Brain Res., 190 (1980) 17-28. 6 Klein, C.M., Guillamondegui, O., Krenek, C.D., LaForte, R.A. and Coggeshall, R.E., Do neuropeptides in the dorsal
154
7
8
9
10
11
horn change if the dorsal root ganglion cell death that normally accompanies peripheral nerve transection is prevented?, Brain Res., 552 (1991) 273-383. Knyihar-Csillik, E., Rakic, P. and Csillik, B., Transganglionic degenerative atrophy in the substantia gelatinosa of the spinal cord after peripheral nerve transection in rhesus monkeys, Cell Tissue Res., 247 (1987) 599-604. McGregor, G.P., Gibson, S.J., Sabate, I.M., Blank, M.A., Christofides, N.D., Wall, P.D., Polak, J.M. and Bloom, S.R., Effect of peripheral nerve section and nerve crush on spinal cord neuropeptides in the rat; increased VIP and PHI in the dorsal horn, Neuroscience, 13 (1984) 207-216. Plenderleith, M.B., Cameron, A.A., Key, B. and Snow, P.J., Soybean agglutinin binds to a subpopulation of primary sensory neurons in the cat, Neurosci. Lett., 86 (1988) 257-262. Plenderleith, M.B., Cameron, A.A., Key, B. and Snow, P.J., The plant lectin soybean agglutinin binds to the soma, axon and terminals of a subpopulation of small-diameter primary sensory neurons in the rat and cat, Neuroscience, 31 (1989) 683-695. Regan, L.J., Dodd, J., Barondes, S.H. and Jessell, T.M., Selective expression of lactose-binding lectins and lactoseries gly-
12
13
14
15
coconjugates in subsets of rat sensory neurons, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 2248-2252. Shehab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy, Exp. Brain Res., 62 (1986) 422-430. Swett, J.E. and Woolf, C.J., The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord, J. Comp. Neurol., 231 (1985) 66-77.11. Villar, M.J., Cortes, R., Theodorsson, E., Wiesenfeld-Hallin, Z., Schalling, M., Fahrenkrug, J., Emson, P.C. and H/)kfelt, T., Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin, Neuroscience, 33 (1989) 587-604. Woolf, C.J., Reynolds, M.L., Molander, C., O'Brien, C., Lindsay, R.M. and Benowitz, L.I., The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury, Neuroscience, 34 (1990) 465-478.
