spinal neurons mediate return of substance p following deafferentation of cat spinal cord

Brain Research, 230 (1981) 263-281 263 Elsevier/North-Holland Biomedical Press

SPINAL NEURONS MEDIATE RETURN OF SUBSTANCE P FOLLOWING DEAFFERENTATION OF CAT SPINAL CORD

A. TESSLER, B. T. HIMES, R. A R T Y M Y S H Y N , M. M U R R A Y and M. E. G O L D B E R G E R

Department of Neurology and Anatomy, VA Medical Center and The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, PA 19129 (U.S.A.)

(Accepted May 5th, 1981)

Key words: substance P - - interneurons - - immunocytochemistry - - deafferentation - - kainic acid - - dorsal horn - - recovery - - cat - - sprouting

S U M M A R Y

Deafferentation of the cat dorsal horn by complete unilateral lumbosacral dorsal rhizotomy ploduces a loss and subsequent partial recovery of substance P (SP) immunoreactivity as visualized by the peroxidase-antiperoxidase technique 3s. The present experiments aimed to determine whether this return of SP represents a generalized response of all fiber systems afferent to the denervated segments or a more selective response of a specific spinal system. Although a contribution from other sources cannot be excluded by this qualitative immunocytochemical technique, several observations indicate that the return of SP staining depends on interneurons which contain SP immunoreactivity: (1) the amount of SP staining in the chronically deafferented dorsal horn deprived of extrinsic fiber systems is comparable to that seen after deafferentation alone; (2) SP-containing neurons are present within the lumbar segments; and (3) destruction of lumbar neurons by the intraspinal injection of kainic acid abolishes SP staining from the chronically deafferented dorsal horn. From these observations it would appear that the anatomical plasticity of SPocontaining fibers in the deafferented dorsal horn is due to the response of a particular system rather than to a generalized response of all systems which terminate there.

I N T R O D U C T I O N

The observed responses to partial denervation of the spinal cord include a depression and partial recovery of function 10,11,30 and an associated loss of afferent

0006-8993/81/0000-0000/$02.75 © Elsevier/North-Holland Biomedical Press

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input followed by partial recoveryl°, 2°. Following deafferentation by dorsal rhizotomy, degeneration staining methods reveal that the projection of some of the undamaged pathways to the denervated segments increases8, 9. This increase may be due to sprouting of the terminals of these pathways. However, it is not known whether the process of recovery is selective, involving a single pathway, or diffuse, involving all pathways afferent to the denervated area. Degeneration methods do not permit an analysis of the response of individual systems to deafferentation. Methods which identify axons according to putative transmitter provide an alternative approach. An immunocytochemical technique has been used to demonstrate that after complete lumbosacral rhizotomy there is a loss and subsequent recovery of substance P (SP) immunoreactivity 38. Neither the disappearance of SP immunoreactivity by 10 days after rhizotomy nor its later recovery is complete. These observations suggest that another SP-containing pathway which overlaps the dorsal root SP projection increases in amount or distribution in response to partial denervation. Because a number of pathways afferent to the denervated region exist, including supraspinal pathways, contralateral dorsal roots, intersegmental propriospinals, and local interneurons, and because SP has been associated with several of these pathways 29, this technique provides a way of determining whether the recovery of SP is due to a generalized response of all the remaining systems which project to the denervated segments or to a more exclusive response by a particular pathway.

The present experiments are designed to determine which spinal system(s) are responsible for the return of SP staining observed in the chronically deafferented dorsal horn. The plan of the experiment is to combine unilateral lumbosacral dorsal root section with an additional lesion which destroys one other potential source of SP immunoreactivity. If SP immunoreactivity returns in spite of the additional lesion, then it must not depend on the lesioned fibers.

MATERIALS AND METHODS

Surgical procedures Twenty cats weighing 3.5-5 kg were used in these experiments. All surgical

procedures were performed under Nembutal anesthesia (35 mg/kg).

(a) Extrinsic sources Here and elsewhere in this report the term 'extrinsic' refers to neuronal systems

which terminate in the lumbar segments but whose perikarya are either not located within the spinal cord or, if within the spinal cord, are distant ( ~ 2 segments) from the segment being studied. In order to evaluate such pathways as sources for the return of SP immunoreactivity to the chronically deafferented dorsal horn, the following procedures were carried out (Figs. 1, 5 and 7).

(1) Spinal cord transection (Fig. 1C, D). Three cats underwent complete unilateral lumbosacral dorsal rhizotomy as previously described zs, survived for one month, and then had spinal cord transection at T13-L1 (two cats; Fig. IC) or L4 (one cat; Fig. 1 D). They were sacrificed by vascular perfusion 11 days after spinal cord transection.

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High lumbar transection permitted evaluation of the contribution made to the restored SP staining by long descending pathways; mid-lumbar (L4) transection, the contribution of both short ascending (in L3) and descending (in L5) propriospinal fibers.

(2) Rhizotomies and ganglionectomies (Fig. 1A, B). In order to evaluate the role of contralateral dorsal afferents, one cat underwent bilateral lumbosacral dorsal rhizotomy (Fig. 1A). In order to evaluate afferents coursing in the ventral roots, one cat had unilateral lumbosacral ganglionectomy (Fig. IB), and one cat had unilateral lumbosacral dorsal and ventral rhizotomy (Fig. 1B). These 3 cats were sacrificed after one month. Because the results of all 3 of these procedures were consistent, only one cat was used for each. The technique used for ganglionectomy has been described s, and the same surgical approach was used for combined dorsal and ventral rhizotomy.

(3) Isolated hernisegment (Fig. 5) and isolated dorsal horn (Fig. 7). In order to determine whether SP immunoreactivity returned to the dorsal horn deprived of all known extrinsic sources, two additional preparations were studied. Three cats underwent a procedure in which right-sided lumbosacral dorsal rhizotomy was performed at the same time as right-sided spinal cord hemisections at L4 and L7 and midline myelotomy (Fig. 5). This preparation is referred to as an isolated hemisegment. An additional 3 cats had right-sided lumbosacral dorsal rhizotomy performed at the same time as ipsilateral hemisection at L4 and undercutting of the right dorsal horn at L4 and below (Fig. 7). The undercutting served to separate the dorsal horn from the intermediate zone. All 6 of these cats were sacrificed after one month. The technique of hemisection has been described 24. Midline myelotomy and dorsal horn undercutting were performed using a shard broken from a razor blade. The incision for undercutting the dorsal horn began at the dentate ligament and angled medially and dorsally.

(b) Intrinsic fibers (1) SP-containing cell bodies. If the recovery of SP immunoreactivity in the

chronically deafferented dorsal horn depends on a local neuronal system, then irnmunoreactive perikarya may be demonstrable within the lumbar segments. There- fore, the distribution of SP-containing cell bodies was examined in sections taken from the L6 segments of 3 normal cats and also in lumbar segmeilts of an additional 3 normal cats pretleated with colchicine. Colchicine aids in the visualization of immuno- reactive perikarya through a mechanism which is thought to involve inhibition of axonal transport 15. A pledget of gelfoam saturated with colchicine (20 mg/ml) remained on the dorsal surface of segments L4-6 for 2 h, and was removed before the wound was closed. The animals were sacrificed 48 h later.

(2) Intraspinal kainic acid. If the return of SP reaction product depends on the presence of interneurons, then destruction of interneurons ought to reduce the antici- pated amounts of staining in the chronically deafferented dorsal horn. Destruction of interneurons on the side with intact dorsal roots should produce little or no reduction, because dorsal roots are the principal source of SP staining in the intact dorsal horn (ref. 29). In order to test this hypothesis the spinal cords of cats which had undergone

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only unilateral lumbosacral dorsal root section 1 month previously were injected on both the intact and deafferented sides with 5 /zg kainic acid, dissolved in 1.0 ,ul of normal saline pH 7.2 (2 cats), and sacrificed 11 days later. 5 injections were made on both sides, approximately 1 cm apart. Kainic acid was injected rapidly into the dorsal horn through a glass pipette (outside tip diameter averaging 0.1 mm) using an air pressure system. The pipette was introduced through the ventrolateral aspect of the spinal cord in order to minimize damage caused by the pipette alone, and the injection was observed under an operating microscope.

Tissue preparation

Cats were anesthetized with Nembutal (35 mg/kg) and perfused through the heart with 4 ~ paraformaldehyde in 0.1 M phosphate buffer pH 7.4. Sections were prepared for the immunocytochemical labeling procedure and for examination by light microscopy, as previously described 3s. The following sections were routinely prepared from each segment: L1-4 and S1, 6-8 sections from the rostral end of each segment; L5-7, 6-8 sections from the rostral end of each segment and 6-8 sections from the middle portion of each segment. Segments which had been injected with kainic acid were cut in serial section in order to identify the site of injection and to map the extent of tissue damage. Every sixth section was stained with cresyl violet or the Mahon stain (for myelin), and sections were selected for immunocytochemical labeling. Segments which had undergone midline myelotomy or dorsal horn undercutting were also examined histologically in order to map the lesion; selected sections were prepared for immunocytochemistry. All sections selected for immunocytochemistry were studied for the presence and distribution of SP immunoreaction product, and representative sections were drawn or photographed.

Blocks of spinal cord which contained a hemisection were embedded in paraffin, sectioned, and stained with cresyl violet or Mahon stains, and the lesion was reconstructed.

Immunocytochemistry

The preparation and characterization of the antiserum to SP have been previously reported 88. Sections were labeled in test tubes by the peroxidase-antiperoxidase (PAP) method of Sternberger 36,3s. In some runs 0.3 ~ Triton X-100 was added to all washes and serum dilutions in order to increase the penetration of reagents into tissue sectionslL Controls consisted of substituting normal rabbit serum or blocked antiserum to SP for SP antiserum in the staining procedure. Blocked antiserum was produced by adding 25 #g of SP to 1 ml of diluted SP antiserum.

RESULTS

Extrinsic fibers

Fig. 1 summarizes the lesions and the extrinsic fiber systems which they

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destroyed. Fig. 2 illustrates a typical result. This cat underwent unilateral dorsal rhizotomy and high lumbar transection which destroys long descending supra- and propriospinal pathways. At 40 days postdeafferentation the dorsal horn contains SP reaction product in every lamina, but it is most intense in laminae I and II and laterally in the base of the dorsal horn. The staining is finely granular and regular. In its

A

B

C

D

SYSTEM LEVEL FIRST LESION SECOND LESION AFFECTED EXAMINED

L1-$3

Unilateral dorsal rhizotomy

OO

OO

Oil'

L1-$3

Contralateral dorsal rhizotomy

L1-$3

Unilateral ganglionectomy

or ventral rhizotomy

L1

T r ~

L4

Transection

L1-$3

Dorsal roots

L1-$3

Dorsal and ventral roots

Long descending pathwaw

(supra- and propriospinals)

Short ascending and

descending propriospinals

L6 ~ ~

and ~

Fig. 1. Summary of lesions in which unilateral lumbosacral dorsal rhizotomy was combined with destruction of additional fiber systems.

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2 Fig. 2. SP staining in the cat dorsal horn (laminae l-Vl). L6 segment, ~ 145, one month after ipsilateral lumbosacral dorsal rhizotomy (L1 $3) and 11 days after high lumbar transection, showing partial recovery of SP reaction product in dorsal horn. Reaction product is finely granular and regular, densest in laminae I, II and V.

74

Fig. 3. SP staining in the cat dorsal horn (laminae l-Vl). L6 segment, i-/, 34, one month after deafferentation by bilateral lumbosacral dorsal rhizotomy (L1-S3).

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staining characteristics, its distribution, and in the amounts present the SP reaction product resembles that seen in the dorsal horns of cats which have undergone deafferentation alone one month before 8s. Similar SP staining occurs one month after deafferentation in spite of lesions which destroy: (1) contralateral dorsal root afferents (Fig. 3); (2) ventral root afferents; (3) ascending or short descending fibers. Therefore, the results of these experiments suggest that the return of SP reaction product does not depend on: long or short descending pathways, short ascending pathways, contralateral dorsal root afferents, or afferents coursing in the ipsilateral ventral roots.

Considerably more SP reaction product is present in the dorsal horn of all these preparations than in the dorsal horn of cats 10 days after deafferentation (Fig. 4). Compared to the dorsal horn of the unoperated cat or the intact side of the unilaterally deafferented animal, the SP reaction product in the dorsal horn of cats which underwent these combined procedures is reduced in amount and stains more finely and regularly. Its pattern of distribution is the same as in controls.

The isolated hemisegment (Fig. 5) provides a test of the hypothesis that the return of SP immunoreactivity does not depend on any of the known potential extrinsic sources of SP. In this preparation the deafferented half of the L6 segment is isolated from ipsilateral ascending and descending fibers as well as from fibers from the opposite side. After one month of survival, finely granular immunoreaction product is present in the dorsal horn in amounts which are comparable qualitatively to those found after rhizotomy alone (Fig. 6). Similar staining occurs in animals in which isolation of the dorsal horn is obtained by combining unilateral deafferentation with hemisection and a lesion which undercuts the dorsal horn (Fig. 7), provided that the lesion is not so extensive as to destroy all neurons in the dorsal horn. When immunocytochemical staining is performed on sections adjacent to those in which Nissl staining reveals dorsal horn neurons to be absent, little SP reaction product is visualized in the dorsal horn.

In summary, the fact that there are no observable differences after these procedures indicates that the return of SP reaction product following deafferentation does not depend on the presence of any extrinsic fibers. These observations suggest that neurons intrinsic to the deafferented segments are the source for the returning SP i mmunoreactivity. Therefore, additional experiments were undertaken in order: (1) to localize perikarya which contain SP immunoreactivity within the spinal cord; and (2) to determine if SP immunoreactivity could be abolished from the chronically deafferented dorsal horn by the local injection of the neurotoxic agent kainic acid.

Intrinsic fibers

SP-eontaining cell bodies Normal. The laminar distribution of cell bodies which demonstrate SP immuno-

reactivity in the L6 segment of 3 normal, unoperated animals which have not been treated with colchicine is summarized in Fig. 8. A total of 43 cells appear in 109 sections taken from the upper and mid portions of the L6 segment. They are limited to lamina X, and the medial portions of laminae VII and VIII.

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Fig. 4. SP staining in the cat dorsal horn (laminae 1-VI). L6 segment, ~'~ 108, 10 days after unilateral lumbosacral dorsal rhizotomy (L1-S3), SP staining is diminished, but a small amount of reaclion product remains, particularly in lamina I.

Thoracic roots

Lumbar roots

Sacral and

caudal roots

5 ISOLATED HEMISEGMENT

Fig. 5. Diagram of preparation designed to deprive one side of the L6 segment of known potential extrinsic sources of SP. ('isolated hemisegment').

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Fig. 6. SP staining in L6 segment of cat dorsal horn (laminae I-VI), one month after isolation of the right side from known potential extrinsic sources of SP, ×31. Note generous amounts of reaction product in laminae I, II, and V. Normal amounts of SP reaction product are present on the intact side.

(b) After colchicine. Fig. 9 illustrates the distribution of perikarya which contain SP immunoreactivity following the local application of colchicine to the dorsal surface of the lumbar spinal cord. The diagram is a composite based on 8 sections taken

from the L5 segment of one normal cat. Such cell bodies and their processes now appear in every lamina of the dorsal horn as well as in the intermediate gray matter. This distribution presumably reflects in part the diffusion of colchicine (Fig. 10a). The number of cell bodies demonstrable in a section is variable, but the dorsal horn in a single section can contain up to 20. These cell bodies do not have a uniform morphology. SP containing perikarya appear similar in morphology to other cells in the same lamina rather than to each other: those in laminae I I and I I I are generally small and lightly staining and can be round, oval or spindle shaped (Fig. 10d). Included among those in the deeper laminae of the dorsal horn are more darkly staining, larger, oval or polygonal cell bodies (Fig. 10c, e), and in lamina I perikarya which are ovoid or elongated and whose processes run parallel to the arc of the dorsal horn (Fig. 10b). An occasional SP-containing cell body appears lateral to the central gray in the dorsola- teral white matter.

Effects of kainic acid In two cats the effects of kainic acid on both the chronically deafferented and

the contralateral, intact side were studied 11 days after the injection of 5/zg kainic acid

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b .

o !

Fig. 7. Cat L6 dorsal horn one month after unilateral lumbosacral dorsal rhizotomy (L1-S3) combined with ipsilateral hemisection (L4) and dorsal horn undercutting, a: reconstruction of lesion undercutting the dorsal horn. Dark stippling indicates intense gliosis, b: SP staining of undercut dorsal horn, x46.

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8

9

Fig. 8. Laminar distribution of cell bodies containing SP immunoreactivity demonstrable without the use of colchicine. Forty-three cells appear in 109 sections taken from the L6 segments of 3 normal, unoperated animals.

at multiple sites bilaterally. Kainic acid produces an apparently limited focus o f

degeneration on both sides o f the cord. Some sections, more distant f rom the injection

site, appear approximately normal in cytoarchitecture and in SP immunoreactivity.

Others closer to the injection site show gliosis, and in these SP immunoreactivi ty is

reduced to a variable degree.

Fig. 9. Distribution of cell bodies containing SP immunoreactivity following application of colchicine. Perikarya observed in 8 widely separated sections from the L5 segment of one normal cat are represented.

2 7 4

Fig. 10. Cell bodies containing SP immunoreact iv i ty following applicat ion of colchicine. L5 segment . a : several cell bodies are indicated by arrows, x 64. b -d : enlargement of cells in laminae I (b), V (c), and I l l (d) at arrows. B, C, C 230 × ; E 148 × .

275

Fig. 11. Normally afferented (left) side of cat dorsal horn (laminae I-VI) one month after contralateral lumbosacral dorsal rhizotomy (L1-$3) and 11 days after bilateral intraspinal injection of kainic acid. L5. a: SP staining. Amount and distribution of reaction product are similar to staining in dorsal horn of normal cat and intact dorsal horn of contralaterally deafferented cats. 40 ×. b: Nissl stain of adjacent section showing gliosis. 40 x Contour of dorsal horn is preserved, c: detail of preceding section. Region indicated by arrow in b. Note cells which resemble neurons. 540 x ,

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Fig. 12. Deafferented (right) side of cat dorsal horn (laminae I-VI) one month after ipsilateral l um bo- sacral dorsal rhizotomy (L1-S3) and 11 days after bilateral intraspinal injection of kainic acid. L6. a: SP staining. SP immunoreactivity is virtually abolished from the deafferented dorsal horn. 40 ×. b: Nissl stain of adjacent section showing gliosis. Contour of dorsal horn is preserved. 40 x . c: detail of preceding section. Region indicated by arrow in b. 540 <.

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On the injected side with intact dorsal roots, some sections are observed in which Nissl staining reveals that neurons remain in the dorsal horn (Fig. 1 lb, c). Adjacent sections prepared for SP immunocytochemistry contain coarsely granular reaction product in the dorsal horn. The amounts of SP staining are comparable to those present in the dorsal horn of normal cats and in the control dorsal horn of unilaterally deafferented cats (Fig. 1 la). In other groups of adjacent sections neurons are absent and there is a slight reduction of SP immunoreactivity in the dorsal horn.

On the deafferented side, sections occur in which Nissl staining demonstrates gray matter destruction or complete neuron loss with a proliferation of astrocytes and microgliacytes (Fig. 12b, c). In adjacent sections immunocytochemistry reveals that SP immunoreactivity in the dorsal horn is abolished or greatly reduced from that expected after chronic deafferentation alone (Fig. 12a). In other rostral or caudal groups of sections neurons survive, and SP staining appears comparable to that seen after chronic deafferentation. Thus, destruction of interneurons by the intraspinal injection of kainic acid effectively eliminates SP reaction product from the chronically deafferented dorsal horn. This result supports the hypothesis that the return of SP immunoreactivity depends on the presence of interneurons.

DISCUSSION

Following unilateral lumbosacral deafferentation by dorsal rhizotomy, immunocytochemical staining indicates that SP staining in the deafferented cat dorsal horn declines to a minimum over the first 10-11 days and then partially recovers by one month zs. One interpretation of the more prominent staining observed in the chronically deafferented dorsal horn is that it represents a compensatory increase in SP in response to previous loss (i.e. anatomical plasticity). If so, then the return of SP may represent an increased amount of SP immunoreaction product in a constant number of processes, or it may represent a proliferation of processes in response to deafferentation (axonal sprouting). Ultrastructural studies are needed to distinguish between these two mechanisms. Another possible interpretation is that the change after deafferentation is due to shrinkage of the spinal cord as a result of the degeneration and removal of the dorsal root axons. If shrinkage were the sole explanation, then it would be expected that other neuropeptides in the dorsal horn would demonstrate a similar sequence of changes. However, immunocytochemical staining carried out on the same tissue for somatostatin and cholecystokinin, peptides which have also been associated with populations of dorsal root afferent fibers 18, has not demonstrated a decline in staining followed by return, as would be predicted if the SP results were due to shrinkage. Deafferentation produces a small but persistent decline in somatostatin staining (ref. 39), whereas cholecystokinin reaction product is virtually abolished (Tessler et al., unpublished observations). These findings make it unlikely that the return of SP immunoreactivity is due to non-specific changes such as shrinkage.

The present investigation provides evidence that the return of SP reaction product does not depend on normally occurring extrinsic sources, such as ascending, descending, or contralateral pathways. The first observation which supports this

278

conclusion is that the amount of SP immunoreactivity which remains in the deafferented dorsal horn deprived of each extrinsic fiber system or isolated from all known potential SP-containing extrinsic sources is comparable to that seen after deafferentation alone. The methods used, however, are incapable of detecting small differences in the amount of staining present after deafferentation alone or combined with removal of one or more potential sources. Therefore, the present results do not exclude a small contribution by extrinsic fibers to the staining observed following deafferentation alone, but do indicate that such staining does not depend on their presence. Included among the systems of fibers terminating in the dorsal horn which have been postulated to contain SP are: (1) a descending projection originating from perikarya in the raphe nuclei of the medulla, some of which fibers may also contain 5-HT 5,a4, which has been reported by some investigators TM, but not by others 18,35,37 ; (2) fibers ascending in the lumbar cord which originate as collaterals of primary afferent fibers or in second-order neurons 26. The present results suggest that the return of SP reaction product after deafferentation depends on interneurons.

SP-containing cell bodies are demonstrable in lamina X and medial laminae VII and VIII in normal cat spinal cord. SP containing perikarya have been observed in the rat spinal cord only with the use of colchicine a5,21,34 or the intramedullary injection ot SP antibody 4. The local application of colchicine to the cat spinal cord demonstrates perikarya in laminae I-VI of the dorsal horn in addition to those which occur normally. Some of them presumably maintain a limited local projection and corres- pond to interneurons as classically definedV, ~2. Cells in lamina I which have elongated cell bodies and processes which parallel the arc of the gray matter resemble Waldeyer

marginal cells, a heterogeneous group of cells which maintain an axon which projects through one or more long pathways 3, including ascending or descending propriospi- rials 2,2s. Other perikarya deeper in the dorsal horn are large, oval, or polygonal in shape, and also resemble projection neurons 4°. Because the axonal trajectories and terminations of these various cells have not been traced, we cannot determine whether any of them contributes to dorsal horn staining either normally or after deafferentation. It is also unclear whether the restored SP immunoreactivity which we attribute to interneurons represents an expansion of terminals normally overlapping those of the dorsal roots or the formation of a new projection by cells which do not normally do so. Either or both possibilities remain, although it appears to be a common finding that in the adult sprouting enlarges an already existing projection rather than cl eating an aberrant onel°,~7, 31,3s. Thus, the results of the present investigation suggest that after deafferentation, the return of SP reaction product depends on the presence of interneurons which contain SP immunoreactivity. The present results do not rule out the possibility that after both dorsal root and interneuronal sources are removed, other sources might act to restore SP. If so, then this would suggest that interneuron sprouting blocks sprouting from other sources. These questions were not addressed in

the present study. That interneurons are responsible for the SP staining which persists after chronic

deafferentiation receives further support from the finding that their destruction by the intraspinal injection of kainic acid abolishes SP staining from the chronically deaffer-

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ented dorsal horn. Kainic acid has been reported to induce rapidly developing de- generative changes in perikarya and dendrites after intraparenchymal or intraventri- cular injection 25, while preferentially sparing axons of passage and afferent fibers6,19, 22,23,41. It has been shown to destroy SP-containing cell bodies in the rat striatum and

produce a decline in SP immunoreactivity in the substantia nigra 17. In the present material, when cell loss due to kainic acid injection is complete, SP staining is a-

bolished in the deafferented dorsal horn*. When, however, only a few cells remain, the SP staining approximates that seen after deafferentation alone. These results suggest that the intrinsic SP projection is derived from overlapping projections of cells

which are somewhat scattered in the gray matter, but which converge in a particular segment. Generally the same pattern is apparent following the injection of kainic acid

into the side with intact dorsal roots. Here SP immunoreactivity is only slightly reduced in sections in which neurons are absent and preserved when neurons remain. A slight reduction could be due to effects on dorsal root or other afferent fibers or on the dorsal root ganglion cells themselveslg,2a,a3, 41. This pattern is consistent also with

the suggestion that interneurons contribute a small amount to the SP immunoreactivity present normally in the cat lumbar dorsal horn 16. The several lines of evidence described in the present report suggest that this amount increases in the dorsal horn chronically deafferented by dorsal root section.

ACKNOWLEDGEMENTS

We thank Dr. Susan E. Leeman for her gift of substance P antiserum, Dr. A. I. Basbaum for suggesting the kainic acid experiments, and Christine Connery for her expert technical assistance, This research was supported by a grant from the VA Medical Center, Philadelphia, and by N I H Grants NS 14477 and NS 13768.

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