Brain Research, 230 (1981) 263-281
Elsevier/North-Holland Biomedical Press
263
SPINAL NEURONS MEDIATE RETURN OF SUBSTANCE P FOLLOWING
DEAFFERENTATION OF CAT SPINAL CORD
A. TESSLER, B. T. H I M E S , 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 h o r n - - recovery - - cat - - sprouting
SUMMARY
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 immunocytochemicaltechnique, 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.
INTRODUCTION
The observed responses to partial denervation of the spinal cord include a
depression and partial recovery of function10,11,30 and an associated loss of afferent
0006-8993/81/0000-0000/$02.75 © Elsevier/North-Holland Biomedical Press
264
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.
1D). They were sacrificed by vascular perfusion 11 days after spinal cord transection.
265
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. Therefore, 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 immunoreactive 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 anticipated 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
266
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 described3s. 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
267
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
AFFECTED EXAMINED
FIRST LESION
SECONDLESION
L1-$3
L1-$3
L1-$3
Unilateraldorsal
rhizotomy
Contralateraldorsal
rhizotomy
Dorsal roots
L1-$3
L1-$3
Unilateral
ganglionectomy
or
ventral rhizotomy
Dorsal and
ventral roots
L1
Long descending
OO
T r ~
OO
pathwaw
L6
~
and
~
(supra- and
propriospinals)
Oil'
L4
Short ascending
and
Transection
descending
propriospinals
Fig. 1. Summary of lesions in which unilateral lumbosacral dorsal rhizotomy was combined with
destruction of additional fiber systems.
268
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).
269
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
immunoreactivity. 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 immunoreactivity 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.
270
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
ISOLATEDHEMISEGMENT
Fig. 5. Diagram of preparation designed to deprive one side of the L6 segment of known potential
extrinsic sources of SP. ('isolated hemisegment').
271
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 II and III 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 dorsolateral 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
272
.
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.
273
8
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 from the injection
site, appear approximately normal in cytoarchitecture and in SP immunoreactivity.
Others closer to the injection site show gliosis, and in these SP immunoreactivity is
reduced to a variable degree.
9
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.
274
Fig. 10. Cell bodies containing SP immunoreactivity following application of colchicine. L5 s e g m e n t .
a : several cell bodies are indicated by arrows, x 64. b - d : enlargement of cells in laminae I (b), V (c),
a n d 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 ,
276
Fig. 12. Deafferented (right) side of cat dorsal horn (laminae I-VI) one month after ipsilateral l um bosacral 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 <.
277
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. 1lb, 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. 1la). 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 immunocytochemistryreveals 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 correspond 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 propriospirials 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-
279
ented dorsal horn. Kainic acid has been reported to induce rapidly developing degenerative changes in perikarya and dendrites after intraparenchymal or intraventricular 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 abolished 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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