Brain Research, 168 (1979) 247-259 © Elsevier/North-HollandBiomedicalPress
247
SUBSTANCE P: DEPLETION IN THE DORSAL HORN OF RAT SPINAL CORD AFTER SECTION OF THE PERIPHERAL PROCESSES OF PRIMARY SENSORY NEURONS
THOMASJESSELL*,AKINOBUTSUNOO,ICHIRO KANAZAWAand MASANORI OTSUKA Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113 and (LK.) Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, lbaraki-ken (Japan)
(Accepted September 14th, 1978)
SUMMARY The substance P content, glutamic acid decarboxylase and choline acetyltransferase activities and the level of [aH]diprenorphine binding were measured in various regions of the lumbar spinal cord of rats after unilateral section of the sciatic nerve or after dorsal rhizotomy. Sciatic nerve section produced a 75-80 ~ depletion of substance P in the dorsal horn but did not change the substance P content of the ventral horn. The onset of substance P depletion occurred within 7 days and was maintained for 2 months. The substance P content of the dorsal root ganglia and both ~.he peripheral and central branches of primary sensory neurons was also reduced after sciatic nerve section. Glutamic acid decarboxylase and choline acetyltransferase activity were unchanged; however, a small decrease in opiate receptor binding occurred I month after nerve section. Dorsal rhizotomy produced an 80 ~ depletion of substance P in the dorsal horn. In addition, the substance P content of the ventral horn was significantly reduced. Glutamic acid decarboxylase activity in the dorsal horn was unaffected by dorsal rhizotomy whereas opiate receptor binding was reduced by 4 0 ~. From these studies it appears that peripheral nerve injury results in the degeneration of primary sensory neurons which contain and release substance P as neurotransmitter.
INTRODUCTION The p~ptide substance P appears to represent one of the transmitters released from the central terminals of primary sensory neurons in the dorsal horn of the spinal * Present address and address for correspondence:Department of Pharmacology,Harvard Medical School, 250 LongwoodAvenue,Boston, Mass. 02115, U.S.A.
248 cord 15,27,33,a4. The substantia gelatinosa of the dorsal horn, in particular, contains a high concentration of substance p15,16,21,44 and the peptide is located over synaptic vesicles in small diameter afferent terminals a,6,36. Although the highest concentrations of substance P are found in the nerve terminal regions 21, synthesis seems to occur in the neuronal cell bodies located in the dorsal root ganglia and transport to the terminals occurs by axonal flow. In support of this, Takahashi and Otsuka 44 have shown that the ligation of cat dorsal roots leads to an accumulation of substance P distal to the ligated site with a marked depletion in the deafferented dorsal horn. The presence of substance P has also been demonstrated in the peripheral branches of primary sensory neuronsa, 16, although the role of substance P in the periphery is uncertain. The degenerative changes in primary afferent terminals in the dorsal horn following dorsal rhizotomy have been extensively studied 13. More recently, a number of studies have described morphological and histochemical changes in the dorsal horn which suggest that a similar atrophy and degeneration of primary afferent terminals occurs following section of the peripheral processes of these neurons 1°,12,2a,24,32. At present, however, the effects of peripheral nerve injury on the neurotransmitter content of the dorsal horn are unknown. In this paper we present evidence that section of the rat sciatic nerve, as well as dorsal rhizotomy, produces a dramatic depletion of the substance P content of the lumbar spinal cord. We have also examined the effect of sciatic nerve section and dorsal rhizotomy on the levels of opiate receptor binding, glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) activity to provide an index of changes in other neuronal populations present in the spinal cord which may interact with substance P-containing primary afferent neurons. METHODS
Operation and dissection Male albino rats (150-200 g) were anesthetized with ether and the left sciatic nerve was exposed and sectioned close to its emergence from the sciatic foramen. Rats were then allowed to recover for periods of 1-57 days. In 5 rats the left sciatic nerve was exposed but was not sectioned, to provide a control for possible damage to the spinal cord during surgery. In a further 6 rats a lumbar laminectomy was performed and dorsal roots L5 and L6 were sectioned unilaterally under pentobarbitone anesthesia, and animals were allowed to recover for 22-28 days. At 1, 2, 4, 7, 13, 22, 28, 36, 47 and 57 days after surgery, groups of 5 rats were anesthetized with ether and a laminectomy was performed between upper thoracic and lower sacral vertebrae. The spinal cord was then removed with the dorsal and ventral roots attached and placed on ice. The central processes of the sciatic nerve were identified and the zone of entry into the spinal cord (L4-$1) was marked. One centimeter sections of operated and control dorsal roots were then dissected and frozen on dry ice. After removing the dorsal roots, the spinal cord was also frozen on dry ice and 4 cross-sections of the spinal cord (comprising segments L5 and L6) were prepared using single-edged razor blades with a fixed inter-blade distance of 0.8 mm.
249
0.5mm Fig. 1. Fresh unfixed, unstained, 200/~m thick cross-section of rat lumbar spinal cord showing (left) the regions removed for determination of substance P, GAD and CAT in spinal gray matter (see Fig. 5) and (right) the dorsal third of the dorsal horn (with surrounding white matter), the ventral horn and ventral white matter used for assay of substance P, GAD and [aH]diprenorphine binding (see Figs. 2 and 4 and Table I). The dorsal third of the dorsal horn (Fig. 1) was then removed along with the surrounding tract of Lissauer, dorsal and dorsolateral funiculi on both operated and unoperated sides. Reproducible sections of spinal cord were produced with this technique (mean wet weight: 2.43 i 0.07 rag; mean ! S.E.M.; n = 10) and tissue from serial sections was frozen for assay of substance P, [3H]diprenorphine binding, and G A D activity. In addition, dorsal root ganglia and the 5 m m lengths of sciatic nerve immediately proximal and distal to the point of section were removed for assay of substance P on the operated side. Dorsal root ganglia and equivalent lengths of sciatic nerve were also taken from the intact side. During dissection care was taken to ensure that regeneration of the cut ends of the sciatic nerve had not occurred. In 5 animals with postoperative survival times of 47 days a detailed study of the substance P content, G A D and CAT activity in the lumbar spinal cord were performed. Spinal cord sections (200/~m) were cut on a cryostat and the spinal gray matter was dissected in a cold box at - - 2 5 to - - 3 0 °C under a binocular microscope. Tissue samples from corresponding regions of two slices were pooled for assay of substance P and CAT and from 3 slices for assay of G A D .
250
Assay of substance P Substance P was measured by a radiolmmunoassay based on methods described previously21,3L Dorsal horn and sciatic nerve tissue was homogenized at 4 °C in 25 vols of 2N acetic acid, using a dental drill, and the homogenate was then heated to 98 °C in a boiling water bath for 5 min to inactivate peptidases. Tissue samples were then placed on ice for a further 30 min to allow for complete extraction of substance P. In control experiments, recovery of [125I]substance P added to the tissue samples was found to be 88 ~ . Heating for periods in excess of 5 rain did not increase the apparent substance P content of dorsal horn or sciatic nerve tissue, measured by radioimmunoassay. After 30 min the homogenate was centrifuged at 2000 × g for 5 min and the resulting supernatant was lyophilized. Samples were reconstructed in 300 kd barbitone acetate buffer (0.05 M; pH 8.6), centrifuged at 2000 × g to remove insoluble material and the supernatant used for assay of substance P-like immunoreactivity using a radioimmunoassay with a sensitivity of 15-20 fmol substance p21.
Assay of glutamic acid decarboxylase activity GAD activity was assayed using a modification~° of the method of Molinoff and KravitzZL
Opiate receptor binding assay To measure opiate receptor binding in very small amounts of dorsal horn tissue, a modification of previously published methods was used aI,26. Frozen tissue samples (3-4 mg wet weight) were homogenized in 360/tl Tris.HCl buffer (0.05 M, pH 7.4) containing 0.1 M sodium chloride at 4 °C. Aliquots (160 #1) were preincubated for 30 min at 30 °C to destroy endogenous opiate receptor ligands and then incubated in a final volume of 200 #1 with 5 nM [aH]diprenorphine (6.l Ci/mmol, Radiochemical Centre, Amersham) for 30 min at 30 °C. For each sample, parallel incubations in the presence and absence of naloxone (5× 10-TM) were performed. Incubation was terminated by centrifugation at 3500 × g for 90 sec followed by removal of the supernatant. The pellet was then resuspended in 200 #1 of distilled water and transferred to a scintillation vial containing I0 ml Instagel (Packard Co.) and counted by liquid scintillation spectrophotometry at 43 ~o efficiency for 10 min. Naloxonedisplaceable binding represented 30-40 ~ of total [3H]diprenorphine binding. Specific binding increased linearly with tissue concentration over the range l-5 rag.
Choline acetyltransferase activity CAT activity was measured using the method of Fonnum 7.
Protein In all assays protein determinations were carried out in each tissue sample by the method of Lowry et al. 29. The protein content of sciatic nerve and dorsal horn tissue was not significantly altered by sciatic nerve section or dorsal rhizotomy.
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Fig. 2. Effect of sciatic nerve section on the substance P content of rat dorsal horn. Each point represents the mean substance P content in fmoi/mg protein (:~ S.E.M.) from 5 animals. Closed circles: unoperated side; open circles: sectioned side. 0, P < 0.05; "k, P < 0.01 (the difference between all operated and unoperated values from day 13 onwards are significant at P < 0.01). Paired t-test. RESULTS The effect o f sciatic nerve section on the substance P content o f rat dorsal horn At 2 and 4 days after unilateral section of the sciatic nerve, the substance P content of the ipsilateral dorsal horn was not significantly different from the control, unoperated side (Fig. 2). From 7 days postoperatively there was a progressive decrease in the substance P content of the dorsal horn until, at 28 days, the operated dorsal horn contained only 20-25 ~o of the substance P content of the control side. At longer postoperative intervals (up to 57 days) no further decrease was observed (Fig. 2). In sham-operated animals there was no significant difference between the substance P content of the dorsal horn on the operated (2898 -q- 245 fmol/mg protein; mean ± S.E.M.; n----5) and control (2589 :[: 255 fmol/mg protein; mean ± S.E.M.; n = 5 ) sides. The effect o f sciatic nerve section on the substance P content o f rat sciatic nerve, dorsal root ganglia and dorsal roots (a) Dorsal roots. The substance P content of the dorsal roots (L~-Le) representing the central processes of the rat sciatic nerve was in the range 100-140 fmol/mg protein, which agrees reasonably well with estimates of the substance P content of bovine and feline dorsal roots4Z, 44. After sciatic nerve section there was a marked decrease in the substance P content of the dorsal roots which preceded the substance P depletion in the dorsal horn (Fig. 3A). By 7 days after surgery the substance P content of the dorsal roots on the operated side was 51 ~ of that on the control side, and by 13 days the substance P content was reduced to 31%. At postoperative times of greater than 13 days there was no additional loss of substance P on the operated side. In shamoperated animals no significant difference was observed in the substance P content of dorsal roots on the operated and control sides.
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days Fig. 3. Effect of sciatic nerve section on the substance P content of rat dorsal roots, dorsal root ganglia and sciatic nerve proximal and distal to the section. Each point represents the mean substance P content in fmol/mg protein ( ± S.E.M.) for 5 animals. Closed circles: unoperated side; open circles: operated side. "A',P < 0.05; ÷ , P < 0.01. Paired t-test. A: dorsal roots; B: dorsal root ganglion; C: sciatic nerve proximal to section; D: sciatic nerve distal to section.
(b) Dorsal root ganglia. The effects of sciatic nerve section on the substance P content of the ipsilateral dorsal root ganglia were complex (Fig. 3B). At 4 and 28 days there was a 47-49 ~ decrease in the substance P content of the dorsal root ganglia (Ls-L6), which paralleled the substance P depletion found in the dorsal horn and dorsal roots. At postoperative times greater than 28 days, however, the difference between operated and unoperated sides was reduced although not completely abolished. (c) Proximal portion of the sectioned sciatic nerve (in uninterrupted contact with the cell body). One day after sciatic nerve section there was a 4.6-fold increase in the substance P content of the 5 mm section of sciatic nerve immediately proximal to the section (Fig. 3C). This increase almost certainly reflects the axonal transport of substance P from cell bodies in the dorsal root ganglia and subsequent accumulation at the site of section, and confirms previous immunohistochemical findings 16. At longer postoperative times, however, there was a marked and prolonged depletion of substance P in the portion of sciatic nerve between the cell bodies and point of section. At 4 days the substance P content was 43 ~ of the equivalent section of contralateral unsectioned nerve, and from 7 to 57 days the depletion was maintained ( 1 5 - 5 2 ~ of control side). There was no difference in the substance P content of operated and unoperated nerves in sham-operated animals.
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Fig. 4. Effect of sciatic nerve section on (A) GAD activity ~mol/mg protein/h) (n = 5 animals) and (B) [aH]diprenorphine binding (fmol/mg protein) (n = 4 or 5 animals) in rat dorsal horn. Closed circles: unoperated side; open circles: operated side. 0, P < 0.1 ; ~ , P < 0.05. Paired t-test.
(d) Distal portion of the sectioned sciatic nerve. One day after sciatic nerve section the substance P content of the distal stump of the sciatic nerve was 41 ~o of the corresponding unoperated section (Fig. 3D). At subsequent times (4--57 days) there was a large (71-96 ~o) and maintained depletion of substance P in the distal stump of the sectioned nerve. Effect of sciatic nerve section on GAD activity in dorsal horn At all postoperative survival times examined (2-57 days) there was no significant difference in the G A D activity in dorsal horn tissue on operated and control sides (Fig. 4A). Effect o f sciatic nerve section on [aH]diprenorphine binding in rat dorsal horn The naloxone-displaceable binding of [aH]diprenorphine to rat dorsal horn tissue ranged between 80 and 120 fmol/mg protein (Fig. 4B). Sciatic nerve section did not produce any effect on [aH]diprenorphine binding at postoperative times of up to 22 days (Fig. 4B). At times greater than 28 days there was a small but significant (P < 0.05) decrease in [aH]diprenorphine binding to dorsal horn tissue on the operated side•
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Fig. 5. Distribution of (A) substance P (fmol/mg protein); (B) GAD ~mol/mg protein/h); and (C) CAT (nmol/mg protein/h) in rat lumbar spinal cord gray matter and the effect of sciatic nerve section. Left: operated side; right: contralateral, unoperated side. Each value is the mean (& S.E.M.) for 3 animals. ~t', P < 0.05. Paired t-test. (47 days postoperative.)
Effect of sciatic nerve section on substance P, GAD and C A T in discrete regions of lumbar spinal cord In 3 animals with sciatic nerve section (47 days postoperative) a detailed mapping of the substance P content, G A D and CAT activity was performed in the lumbar spinal gray matter. Following sciatic nerve section there was a 74 % depletion in the substance P content of the most superficial laminae (I-III) of the dorsal horn (Fig. 5A), however, a smaller (23 ~ ) but significant (P < 0.05) depletion of substance P also occurred in laminae IV and V. The substance P content of the intermediate zone and ventral horn was not significantly different on lesioned and control sides (Fig. 5A). G A D activity was evenly distributed in the dorsal and intermediate zones (Fig. 5B) but was somewhat lower in the ventral horn. The distribution of G A D , therefore, differs from that of substance P in the rat spinal cord. Furthermore, G A D appears to be more evenly distributed than G A B A in mammalian spinal cord a°. Sciatic nerve
255 TABLE I Effect of dorsal rhizotomy on (A) substance P content of peripheral and central regions of primary sensory neurons of the rat; (13) [aH]diprenorphine binding in dorsal horn (fmol/mg protein); and (C) GAD activity~mol/mg protein/h) from 6 animals (survival time 22-28 days), q- P < 0.01 ; *P < 0.05. Paired t-test.
Control (A) Substance P: fmol/mg protein Dorsal horn 2759 4- 216 Ventral horn 695 4- 46 Ventral white 31 4- 10 Dorsal root (prox.) 113 4- 26 Dorsal root (dist.) 84 4- 18 Dorsal root ganglia 213 4- 40 Sciatic nerve 91 ± 16
Rhizotomy 559 4- 24** 388 4- 31 ** 41 4- 12 26 4- 12"* 157 4- 29* 268 4- 13 98 4- 8
(B) [aH]Diprenorphine binding: fmol/mg protein 78 4- 10
41 4-9*
(C) GAD activity:/~mol/mg protein/h Dorsal horn 409 4- 15
427 4- 22
Dorsal horn
* P < 0.05; ** P < 0.01; paired t-test.
section produced no significant change in G A D activity in any region of lumbar spinal gray matter. CAT activity in rat lumbar spinal cord was much higher in the ventral horn than in the intermediate and dorsal gray matter (Fig. 5C), which corresponds with the distribution of motoneurone cell bodies. Sciatic nerve section did not alter CAT activity in any region of lumbar spinal cord. The activity of CAT in motoneurone cell bodies, therefore, probably was not affected by section of the motoneuron axon.
The effect of dorsal rhizotomy on substance P content, GAD activity and [aHjdiprenorphine binding in the rat dorsal horn In 6 rats with unilateral section of L5 and Le dorsal roots (22-28 days postoperative) the substance P content in the dorsal horn of the operated side was reduced to 20 % of that of the unoperated dorsal horn (Table IA). There was also a 44 ~o decrease in the substance P content of the ventral horn on the operated side while the substance P content of the ventral white matter was unchanged. In the dorsal roots distal to the section there was a 1.87-fold increase in the substance P content compared to the equivalent length of contralateral unoperated dorsal root. In dorsal roots proximal to the section the substance P content was reduced to 23% of the corresponding, unoperated dorsal roots. The substance P content of the dorsal root ganglia and the peripheral part of the sciatic nerve was not significantly changed by dorsal rhizotomy (Table IA). In the lumbar dorsal horn of the same animals there was a 47 ~ decrease in [aH]diprenorphine binding (Table IB) while there was no significant decrease in G A D activity (Table IC).
256 DISCUSSION It has been known for many years that injury to the axon of a neuron results in morphological changes in the neuronal perikarya in addition to Wallerian degeneration in the severed axonal stump (see Lieberman 2s for a review). In primary sensory neurons, which exhibit a pronounced functional and anatomical polarization of their neuronal processes, section of the peripheral axon results in a marked cell loss in spinal 2,~a,as and trigeminal 1,s ganglia which appears to be largely restricted to small neurons 1,~,as. Similar degenerative changes have been reported in the central axon branches and terminals of primary sensory neurons l°,12,2a,24,a2. It is likely, therefore, that the profound depletion of substance P in the dorsal horn and spinal roots following peripheral nerve transection reflects the degeneration of substance Pcontaining primary sensory neurons. In support of this, the time course of substance P depletion appears to parallel the degenerative changes observed in primary afferent terminals13,1s, 23. It is unclear at present whether cell loss occurs exclusively in primary sensory neurons that contain substance P. Some evidence exists for the degeneration of a significant number of larger diameter neurons 1; whether any larger diameter sensory neurons contain substance P remains to be established. There appear to be a number of important differences in the effects of sciatic nerve section and dorsal rhizotomy on the substance P content of the various structural components of primary sensory neurons. Dorsal rhizotomy did not alter the substance P content of the sciatic nerve, which suggests that the transport of substance P from cell bodies to the periphery was unaffected. In contrast, sciatic nerve section produced a depletion of substance P in all parts of the primary sensory neuron, including the cell bodies. These results parallel morphological studies in which chromatolysis and degeneration of dorsal root ganglion cells occurs after peripheral nerve section but not after dorsal rhizotomy 4. In the spinal cord the effect of sciatic nerve section appeared to be confined to the dorsal horn whereas dorsal rhizotomy also produced a significant decrease in substance P in the ventral horn. It is unlikely that the decrease in the substance P content of the ventral horn resulted from non-specific damage since in this case a decrease in the G A D activity in the spinal cord would have been expected. In previous studies Takahashi and Otsuka aa have demonstrated a similar decrease in the substance P content of the cat ventral horn after dorsal rhizotomy. Substance P may, therefore, be contained in some primary afferent fibers which project to the ventral horn. It is also possible that the transsynaptic degeneration of substance P-containing interneurons which originate in the spinal cord could account for the decrease in substance P. H6kfelt et al. 16,17 have described the presence of scattered substance P-positive cell bodies in the spinal cord. It is clear that the origin of substance P in the ventral horn requires a more detailed examination. Morphological studies of synaptic glomeruli in the substantia gelatinosa of the dorsal horn and spinal trigeminal nucleus have shown that the terminals of small diameter afferent fibers receive axoaxonic synapses from two or more classes of substantia gelatinosa interneuronsg,aL The most likely candidates as transmitter
257 substances released by these interneurons are GABA and the opioid peptide enkephalin, both of which are present in a high concentration in dorsal horn interneurons 17,8°,40,al. In the present experiments the decrease in substance P and opiate receptor binding after sciatic nerve section and dorsal rhizotomy provides further evidence that opiate receptors may be located on the terminals of substance Pcontaining primary afferent neurons in the substantia gelatinosa19,26,4L However, the incomplete loss of opiate receptor binding after both operative procedures suggests that only one population of opiate receptors in the dorsal horn are located on primary afferent terminals. G A D activity in the dorsal horn was unchanged by dorsal rhizotomy which is consistent with previous studies showing that the GABA concentration in the dorsal horn is also unchanged 4°,44. In the rat cervical spinal cord, however, a significant decrease in G A D has been reported after dorsal rhizotomyZ2; the reason for the discrepancy between these results is not clear. In the present experiments, the depletion of substance P in the dorsal horn after sciatic nerve section was maintained for up to two months postoperatively. Knyih~ir and Csillik 2z,24 have provided some anatomical evidence that at longer recovery periods after sciatic nerve section, those primary sensory neurons undergoing only partial degeneration may re-establish synaptic contact with second order neurons in the dorsal horn. It would be interesting, therefore, to examine whether substance P levels return to normal with longer recovery periods indicating partial regeneration or possibly the sprouting of surviving neurons. In addition it remains to be established whether neurons in the dorsal horn which respond to both nociceptive stimuli and substance p14,87 exhibit changes in sensitivity to substance P after peripheral nerve injuries, as appears to be the case following dorsal rhizotomy4L ACKNOWLEDGEMENTS Part of this work was supported by research grants from the Japanese Ministry of Education. T.M.J. was supported by a Royal Society Japan Programme Fellowship. We thank Miss H. Taira and Mr. D. Sutoo for excellent technical assistance.
REFERENCES 1 Aldskoglus, H. and Arvidsson, J., Nerve cell degeneration and death in the trigeminal ganglion of the adult rat following peripheral nerve transection, J. Neurocytol., 7 (1978) 229-250. 2 Cavanaugh, M. W., Quantitative effects of the peripheral innervation area on nerves and spinal ganglion cells, J. comp. Neurol., 94 (1951) 181-219. 3 Chan-Palay, V. and Palay, S. L., Ultrastructural identification of substance P cellsand their processes in rat sensory ganglia and their terminals in the spinal cord by immunocytochemistry,Prec. nat. Acad. Sci. (Wash.), 74 (1977) 4050-4054. 4 Cragg, B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 5 Cuello, A. C., Del Fiacco, M. and Paxinos, G., The central and peripheral ends of the substance Pcontaining sensory neurones in the rat trigcminal system, Brain Research, 152 (1978) 499-509. 6 Cuello, A. C., Jessell, T. M., Kanazawa, I. and Iversen, L. L., Substance P: localization in synaptic vesiclesin rat central nervous system, J. Neurochem., 29 (1977) 747-751. 7 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409.
258 8 Galich, J. W., Gregg, J. M. and Duncan, G. H., Quantitative study of trigeminal ganglion response to peripheral nerve trauma, J. Dental Res., 55 (1976) B109. 9 Gobel, S., Golgi studies of the substantia gelatinosa neurons in the spinal trigeminal nucleus, J. comp. Neurol., 162 (1975) 397-416. 10 Gobel, S. and Binck, J. M., Degenerative changes in primary trigeminal axons and in neurons in nucleus caudalis following tooth pulp extirpations in the cat, Brain Research, 132 (1977) 347-354. 11 Goldstein, A., Lowney, L. I. and Pal, B. K., Stereospecific and non-specific interactions of the morphine congener levorphanol in subcellular fractions of mouse brain, Proc. nat. A cad. Sci. (Wash.), 68 (1971) 1742-1747. 12 Goode, G. E., The ultrastructural identification of primary and suprasegrnental afferents in the marginal and gelatinous layers of lumbar spinal cord following central and peripheral lesions, Soc. Neurosci. Abstr., 11, Part 2 (1976) 975. 13 Heimer, L. and Wall, P. D., The dorsal root distribution to the substantia gelatinosa of the rat with a note on the distribution in the cat, Exp. Brain Res., 6 (1968) 89-99. 14 Henry• J. L.• E•ects •f substance P •n functi•na••y identi•ed units in cat spina• c•rd• Brain Resear ch• 114 (1976) 439-451. 15 Htikfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurones, Brain Research, 100 (1975) 235-252. 16 H6kfelt, T., Johansson, O., Kellerth, J. O., Ljundahl, A., Nilsson, G., Nyg~trds,A. and Pernow, B., Immunohistochemical distribution of substance P. In U. S. von Euter and B. Pernow (Eds.), Substance P, Raven Press, New York, 1977, pp. 117-145. 17 H/Skfelt, T. Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemicalanalysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. nat. Acad. Sci. (Wash..), 74 (1977) 3081-3085. 18 Humbertson, A., A chronological study of the degenerative phenomena of dorsal root ganglia cells following section of the sciatic nerve, Anat. Rec., 145 (1963) 244. 19 Jessell, T. M. and Iversen, L. L., Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature (Land.), 268 (1977) 549-551. 20 Kanazawa, I., Iversen, L. L. and Kelly, J. S., Glutamate decarboxylase activity in the rat posterior pituitary, pineal gland, dorsal root ganglion and superior cervical ganglion, J. Neurochem., 27 (1976) 1267-1269. 21 Kanazawa, I. and Jessell, T. M., Post-mortem changes and regional distribution of substance P in the rat and mouse nervous system, Brain Research, 117 (1976) 362-367. 22 Kelly, J. S., Gottesfeld, Z. and Schon, F., Reduction in GAD 1 activity from the dorsal lateral region of the deafferented rat spinal cord, Brain Research, 62 (1973) 581-586. 23 Knyihfir, E. and Csillik, B., Effect of peripheral axotomy on the fine structure and histochemistry of the Rolando substance: degenerative atrophy of central processes of pseudounipolar cells, Exp. Brain Res., 26 (1976) 73-87. 24 Knyihfir, E. and Csillik, B., Representation of cutaneous afferents by fluoride-resistant acid phosphatase (FRAP)-active terminals in the rat substantia gelatinosa Rolandi, Acta neurol, scand., 53 (1976) 217-225. 25 Knyihgtr, E., Lfiszl6, I. and Tornyos, S., Fine structure and fluoride-resistant acid phosphatase activity of electron dense sinusoid terminals in the substantia gelatinosa Rolandi of the rat after dorsal root transection, Exp. Brain Res., 19 (1974) 529-544. 26 Lamotte, C., Pert, C. B. and Snyder, S. H., Opiate receptor bindingin primate spinal cord: distribution and changes after dorsal root section, Brain Research, 112 (1976) 407-412. 27 Lembeck, F., Zur Frage der zentralen Ubertragung afferenter Impulse. III. Das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Riickenmarks, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 219 (1953) 197-213. 28 Lieberman, A. R., The axon reaction: a review of the principal features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 29 Lowry, O. H., Rosenbrough, N. J., Farr, A, L. and Randall, R. J., Protein measurement with the Folin phenol reagent. J. biol. Chem., 193 (1951) 265-275. 30 Miyata, Y. and Otsuka, M., Quantitative histochemistry of ~,-aminobutyric acid in cat spinal cord with special reference to presynaptic inhibition,J. Neurochem., 25 (1975) 239-244. 31 Molinoff, P. B. and Kravitz, E. A., The metabolism of T-aminobutyricacid (GABA) in the lobster nervous system - - glutamic decarboxylase, J. Neurochem., 15 (1968) 391-409.
259 32 Moradian, G. P. and Rustioni, A., Transganglionic degeneration in the dorsal horn and dorsal column nucleii of adult rats, Anat. Rec., 187 (1977) 660. 33 Otsuka, M. and Konishi, S., Substance P and excitatory transmitter of primary sensory neurons, Cold Spr. Harb. Syrup. quant. Biol., 40 (1975) 135-143. 34 Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat, Nature (Lond.), 264 (1976) 83-84. 35 Powell, D., Leoman, S. E., Tregear, G. W., Niall, H. D. and Potts, J. T., Jr., Radioimmunoassayfor substance P, Nature New Biol., 241 (1973) 252-254. 36 Pickel, V., Reis, D. J. and Leeman, S. E., Ultrastructural localization of substance P in neurons of rat spinal cord, Brain Research, 122 (1977) 534-540. 37 Randi~, M. and Mileti~, V., Effects of substance P in cat dorsal horn neurons activated by noxious stimuli, Brain Research, 128 (1977) 164-169. 38 Ranson, S. W., Retrograde degeneration in the spinal nerves, J. comp. Neurol., 16 (1906) 265-293. 39 R6thelyi, M. and Szent~tgothai,J., Distributionand connections of afferent fibers in the spinal cord. In A. Iggo (Ed.), Somatosensory Systems, Handbook of Sensory Physiology, Vol. 2, Springer Verlag, Berlin, 1973, pp. 207-270. 40 Roberts, P. J. and Keen, P., Effect of dorsal root section on amino acids of rat spinal cord, Brain Research, 74 (1974) 333-337. 41 Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 2167-2171. 42 Simon, E. J., Hiller, J. M., Crain, S. M. and Peterson, E. R., Demonstration ofstereospecific opiate binding in cultures of fetal mouse dorsal root ganglia and spinal cord, Soc. Neurosci. Abstr., (1977) 301. 43 Takahashi, T., Konishi, S., Powell, D., Leeman, S. E. and Otsuka, M., Identification of the motoneuron-depolarizingpeptide in bovine dorsal root as hypothalamic substance P, Brain Research, 73 (1974) 59-69. 44 Takahashi, T. and Otsuka, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brain Research, 87 (1975) 1-11. 45 Wright, D. M. and Roberts, M. H. T., Supersensitivity to a substance P analogue following dorsal root section, Life Sci., 22 (1978) 19-24.
247
SUBSTANCE P: DEPLETION IN THE DORSAL HORN OF RAT SPINAL CORD AFTER SECTION OF THE PERIPHERAL PROCESSES OF PRIMARY SENSORY NEURONS
THOMASJESSELL*,AKINOBUTSUNOO,ICHIRO KANAZAWAand MASANORI OTSUKA Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113 and (LK.) Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, lbaraki-ken (Japan)
(Accepted September 14th, 1978)
SUMMARY The substance P content, glutamic acid decarboxylase and choline acetyltransferase activities and the level of [aH]diprenorphine binding were measured in various regions of the lumbar spinal cord of rats after unilateral section of the sciatic nerve or after dorsal rhizotomy. Sciatic nerve section produced a 75-80 ~ depletion of substance P in the dorsal horn but did not change the substance P content of the ventral horn. The onset of substance P depletion occurred within 7 days and was maintained for 2 months. The substance P content of the dorsal root ganglia and both ~.he peripheral and central branches of primary sensory neurons was also reduced after sciatic nerve section. Glutamic acid decarboxylase and choline acetyltransferase activity were unchanged; however, a small decrease in opiate receptor binding occurred I month after nerve section. Dorsal rhizotomy produced an 80 ~ depletion of substance P in the dorsal horn. In addition, the substance P content of the ventral horn was significantly reduced. Glutamic acid decarboxylase activity in the dorsal horn was unaffected by dorsal rhizotomy whereas opiate receptor binding was reduced by 4 0 ~. From these studies it appears that peripheral nerve injury results in the degeneration of primary sensory neurons which contain and release substance P as neurotransmitter.
INTRODUCTION The p~ptide substance P appears to represent one of the transmitters released from the central terminals of primary sensory neurons in the dorsal horn of the spinal * Present address and address for correspondence:Department of Pharmacology,Harvard Medical School, 250 LongwoodAvenue,Boston, Mass. 02115, U.S.A.
248 cord 15,27,33,a4. The substantia gelatinosa of the dorsal horn, in particular, contains a high concentration of substance p15,16,21,44 and the peptide is located over synaptic vesicles in small diameter afferent terminals a,6,36. Although the highest concentrations of substance P are found in the nerve terminal regions 21, synthesis seems to occur in the neuronal cell bodies located in the dorsal root ganglia and transport to the terminals occurs by axonal flow. In support of this, Takahashi and Otsuka 44 have shown that the ligation of cat dorsal roots leads to an accumulation of substance P distal to the ligated site with a marked depletion in the deafferented dorsal horn. The presence of substance P has also been demonstrated in the peripheral branches of primary sensory neuronsa, 16, although the role of substance P in the periphery is uncertain. The degenerative changes in primary afferent terminals in the dorsal horn following dorsal rhizotomy have been extensively studied 13. More recently, a number of studies have described morphological and histochemical changes in the dorsal horn which suggest that a similar atrophy and degeneration of primary afferent terminals occurs following section of the peripheral processes of these neurons 1°,12,2a,24,32. At present, however, the effects of peripheral nerve injury on the neurotransmitter content of the dorsal horn are unknown. In this paper we present evidence that section of the rat sciatic nerve, as well as dorsal rhizotomy, produces a dramatic depletion of the substance P content of the lumbar spinal cord. We have also examined the effect of sciatic nerve section and dorsal rhizotomy on the levels of opiate receptor binding, glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) activity to provide an index of changes in other neuronal populations present in the spinal cord which may interact with substance P-containing primary afferent neurons. METHODS
Operation and dissection Male albino rats (150-200 g) were anesthetized with ether and the left sciatic nerve was exposed and sectioned close to its emergence from the sciatic foramen. Rats were then allowed to recover for periods of 1-57 days. In 5 rats the left sciatic nerve was exposed but was not sectioned, to provide a control for possible damage to the spinal cord during surgery. In a further 6 rats a lumbar laminectomy was performed and dorsal roots L5 and L6 were sectioned unilaterally under pentobarbitone anesthesia, and animals were allowed to recover for 22-28 days. At 1, 2, 4, 7, 13, 22, 28, 36, 47 and 57 days after surgery, groups of 5 rats were anesthetized with ether and a laminectomy was performed between upper thoracic and lower sacral vertebrae. The spinal cord was then removed with the dorsal and ventral roots attached and placed on ice. The central processes of the sciatic nerve were identified and the zone of entry into the spinal cord (L4-$1) was marked. One centimeter sections of operated and control dorsal roots were then dissected and frozen on dry ice. After removing the dorsal roots, the spinal cord was also frozen on dry ice and 4 cross-sections of the spinal cord (comprising segments L5 and L6) were prepared using single-edged razor blades with a fixed inter-blade distance of 0.8 mm.
249
0.5mm Fig. 1. Fresh unfixed, unstained, 200/~m thick cross-section of rat lumbar spinal cord showing (left) the regions removed for determination of substance P, GAD and CAT in spinal gray matter (see Fig. 5) and (right) the dorsal third of the dorsal horn (with surrounding white matter), the ventral horn and ventral white matter used for assay of substance P, GAD and [aH]diprenorphine binding (see Figs. 2 and 4 and Table I). The dorsal third of the dorsal horn (Fig. 1) was then removed along with the surrounding tract of Lissauer, dorsal and dorsolateral funiculi on both operated and unoperated sides. Reproducible sections of spinal cord were produced with this technique (mean wet weight: 2.43 i 0.07 rag; mean ! S.E.M.; n = 10) and tissue from serial sections was frozen for assay of substance P, [3H]diprenorphine binding, and G A D activity. In addition, dorsal root ganglia and the 5 m m lengths of sciatic nerve immediately proximal and distal to the point of section were removed for assay of substance P on the operated side. Dorsal root ganglia and equivalent lengths of sciatic nerve were also taken from the intact side. During dissection care was taken to ensure that regeneration of the cut ends of the sciatic nerve had not occurred. In 5 animals with postoperative survival times of 47 days a detailed study of the substance P content, G A D and CAT activity in the lumbar spinal cord were performed. Spinal cord sections (200/~m) were cut on a cryostat and the spinal gray matter was dissected in a cold box at - - 2 5 to - - 3 0 °C under a binocular microscope. Tissue samples from corresponding regions of two slices were pooled for assay of substance P and CAT and from 3 slices for assay of G A D .
250
Assay of substance P Substance P was measured by a radiolmmunoassay based on methods described previously21,3L Dorsal horn and sciatic nerve tissue was homogenized at 4 °C in 25 vols of 2N acetic acid, using a dental drill, and the homogenate was then heated to 98 °C in a boiling water bath for 5 min to inactivate peptidases. Tissue samples were then placed on ice for a further 30 min to allow for complete extraction of substance P. In control experiments, recovery of [125I]substance P added to the tissue samples was found to be 88 ~ . Heating for periods in excess of 5 rain did not increase the apparent substance P content of dorsal horn or sciatic nerve tissue, measured by radioimmunoassay. After 30 min the homogenate was centrifuged at 2000 × g for 5 min and the resulting supernatant was lyophilized. Samples were reconstructed in 300 kd barbitone acetate buffer (0.05 M; pH 8.6), centrifuged at 2000 × g to remove insoluble material and the supernatant used for assay of substance P-like immunoreactivity using a radioimmunoassay with a sensitivity of 15-20 fmol substance p21.
Assay of glutamic acid decarboxylase activity GAD activity was assayed using a modification~° of the method of Molinoff and KravitzZL
Opiate receptor binding assay To measure opiate receptor binding in very small amounts of dorsal horn tissue, a modification of previously published methods was used aI,26. Frozen tissue samples (3-4 mg wet weight) were homogenized in 360/tl Tris.HCl buffer (0.05 M, pH 7.4) containing 0.1 M sodium chloride at 4 °C. Aliquots (160 #1) were preincubated for 30 min at 30 °C to destroy endogenous opiate receptor ligands and then incubated in a final volume of 200 #1 with 5 nM [aH]diprenorphine (6.l Ci/mmol, Radiochemical Centre, Amersham) for 30 min at 30 °C. For each sample, parallel incubations in the presence and absence of naloxone (5× 10-TM) were performed. Incubation was terminated by centrifugation at 3500 × g for 90 sec followed by removal of the supernatant. The pellet was then resuspended in 200 #1 of distilled water and transferred to a scintillation vial containing I0 ml Instagel (Packard Co.) and counted by liquid scintillation spectrophotometry at 43 ~o efficiency for 10 min. Naloxonedisplaceable binding represented 30-40 ~ of total [3H]diprenorphine binding. Specific binding increased linearly with tissue concentration over the range l-5 rag.
Choline acetyltransferase activity CAT activity was measured using the method of Fonnum 7.
Protein In all assays protein determinations were carried out in each tissue sample by the method of Lowry et al. 29. The protein content of sciatic nerve and dorsal horn tissue was not significantly altered by sciatic nerve section or dorsal rhizotomy.
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Fig. 2. Effect of sciatic nerve section on the substance P content of rat dorsal horn. Each point represents the mean substance P content in fmoi/mg protein (:~ S.E.M.) from 5 animals. Closed circles: unoperated side; open circles: sectioned side. 0, P < 0.05; "k, P < 0.01 (the difference between all operated and unoperated values from day 13 onwards are significant at P < 0.01). Paired t-test. RESULTS The effect o f sciatic nerve section on the substance P content o f rat dorsal horn At 2 and 4 days after unilateral section of the sciatic nerve, the substance P content of the ipsilateral dorsal horn was not significantly different from the control, unoperated side (Fig. 2). From 7 days postoperatively there was a progressive decrease in the substance P content of the dorsal horn until, at 28 days, the operated dorsal horn contained only 20-25 ~o of the substance P content of the control side. At longer postoperative intervals (up to 57 days) no further decrease was observed (Fig. 2). In sham-operated animals there was no significant difference between the substance P content of the dorsal horn on the operated (2898 -q- 245 fmol/mg protein; mean ± S.E.M.; n----5) and control (2589 :[: 255 fmol/mg protein; mean ± S.E.M.; n = 5 ) sides. The effect o f sciatic nerve section on the substance P content o f rat sciatic nerve, dorsal root ganglia and dorsal roots (a) Dorsal roots. The substance P content of the dorsal roots (L~-Le) representing the central processes of the rat sciatic nerve was in the range 100-140 fmol/mg protein, which agrees reasonably well with estimates of the substance P content of bovine and feline dorsal roots4Z, 44. After sciatic nerve section there was a marked decrease in the substance P content of the dorsal roots which preceded the substance P depletion in the dorsal horn (Fig. 3A). By 7 days after surgery the substance P content of the dorsal roots on the operated side was 51 ~ of that on the control side, and by 13 days the substance P content was reduced to 31%. At postoperative times of greater than 13 days there was no additional loss of substance P on the operated side. In shamoperated animals no significant difference was observed in the substance P content of dorsal roots on the operated and control sides.
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days Fig. 3. Effect of sciatic nerve section on the substance P content of rat dorsal roots, dorsal root ganglia and sciatic nerve proximal and distal to the section. Each point represents the mean substance P content in fmol/mg protein ( ± S.E.M.) for 5 animals. Closed circles: unoperated side; open circles: operated side. "A',P < 0.05; ÷ , P < 0.01. Paired t-test. A: dorsal roots; B: dorsal root ganglion; C: sciatic nerve proximal to section; D: sciatic nerve distal to section.
(b) Dorsal root ganglia. The effects of sciatic nerve section on the substance P content of the ipsilateral dorsal root ganglia were complex (Fig. 3B). At 4 and 28 days there was a 47-49 ~ decrease in the substance P content of the dorsal root ganglia (Ls-L6), which paralleled the substance P depletion found in the dorsal horn and dorsal roots. At postoperative times greater than 28 days, however, the difference between operated and unoperated sides was reduced although not completely abolished. (c) Proximal portion of the sectioned sciatic nerve (in uninterrupted contact with the cell body). One day after sciatic nerve section there was a 4.6-fold increase in the substance P content of the 5 mm section of sciatic nerve immediately proximal to the section (Fig. 3C). This increase almost certainly reflects the axonal transport of substance P from cell bodies in the dorsal root ganglia and subsequent accumulation at the site of section, and confirms previous immunohistochemical findings 16. At longer postoperative times, however, there was a marked and prolonged depletion of substance P in the portion of sciatic nerve between the cell bodies and point of section. At 4 days the substance P content was 43 ~ of the equivalent section of contralateral unsectioned nerve, and from 7 to 57 days the depletion was maintained ( 1 5 - 5 2 ~ of control side). There was no difference in the substance P content of operated and unoperated nerves in sham-operated animals.
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Fig. 4. Effect of sciatic nerve section on (A) GAD activity ~mol/mg protein/h) (n = 5 animals) and (B) [aH]diprenorphine binding (fmol/mg protein) (n = 4 or 5 animals) in rat dorsal horn. Closed circles: unoperated side; open circles: operated side. 0, P < 0.1 ; ~ , P < 0.05. Paired t-test.
(d) Distal portion of the sectioned sciatic nerve. One day after sciatic nerve section the substance P content of the distal stump of the sciatic nerve was 41 ~o of the corresponding unoperated section (Fig. 3D). At subsequent times (4--57 days) there was a large (71-96 ~o) and maintained depletion of substance P in the distal stump of the sectioned nerve. Effect of sciatic nerve section on GAD activity in dorsal horn At all postoperative survival times examined (2-57 days) there was no significant difference in the G A D activity in dorsal horn tissue on operated and control sides (Fig. 4A). Effect o f sciatic nerve section on [aH]diprenorphine binding in rat dorsal horn The naloxone-displaceable binding of [aH]diprenorphine to rat dorsal horn tissue ranged between 80 and 120 fmol/mg protein (Fig. 4B). Sciatic nerve section did not produce any effect on [aH]diprenorphine binding at postoperative times of up to 22 days (Fig. 4B). At times greater than 28 days there was a small but significant (P < 0.05) decrease in [aH]diprenorphine binding to dorsal horn tissue on the operated side•
254
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Fig. 5. Distribution of (A) substance P (fmol/mg protein); (B) GAD ~mol/mg protein/h); and (C) CAT (nmol/mg protein/h) in rat lumbar spinal cord gray matter and the effect of sciatic nerve section. Left: operated side; right: contralateral, unoperated side. Each value is the mean (& S.E.M.) for 3 animals. ~t', P < 0.05. Paired t-test. (47 days postoperative.)
Effect of sciatic nerve section on substance P, GAD and C A T in discrete regions of lumbar spinal cord In 3 animals with sciatic nerve section (47 days postoperative) a detailed mapping of the substance P content, G A D and CAT activity was performed in the lumbar spinal gray matter. Following sciatic nerve section there was a 74 % depletion in the substance P content of the most superficial laminae (I-III) of the dorsal horn (Fig. 5A), however, a smaller (23 ~ ) but significant (P < 0.05) depletion of substance P also occurred in laminae IV and V. The substance P content of the intermediate zone and ventral horn was not significantly different on lesioned and control sides (Fig. 5A). G A D activity was evenly distributed in the dorsal and intermediate zones (Fig. 5B) but was somewhat lower in the ventral horn. The distribution of G A D , therefore, differs from that of substance P in the rat spinal cord. Furthermore, G A D appears to be more evenly distributed than G A B A in mammalian spinal cord a°. Sciatic nerve
255 TABLE I Effect of dorsal rhizotomy on (A) substance P content of peripheral and central regions of primary sensory neurons of the rat; (13) [aH]diprenorphine binding in dorsal horn (fmol/mg protein); and (C) GAD activity~mol/mg protein/h) from 6 animals (survival time 22-28 days), q- P < 0.01 ; *P < 0.05. Paired t-test.
Control (A) Substance P: fmol/mg protein Dorsal horn 2759 4- 216 Ventral horn 695 4- 46 Ventral white 31 4- 10 Dorsal root (prox.) 113 4- 26 Dorsal root (dist.) 84 4- 18 Dorsal root ganglia 213 4- 40 Sciatic nerve 91 ± 16
Rhizotomy 559 4- 24** 388 4- 31 ** 41 4- 12 26 4- 12"* 157 4- 29* 268 4- 13 98 4- 8
(B) [aH]Diprenorphine binding: fmol/mg protein 78 4- 10
41 4-9*
(C) GAD activity:/~mol/mg protein/h Dorsal horn 409 4- 15
427 4- 22
Dorsal horn
* P < 0.05; ** P < 0.01; paired t-test.
section produced no significant change in G A D activity in any region of lumbar spinal gray matter. CAT activity in rat lumbar spinal cord was much higher in the ventral horn than in the intermediate and dorsal gray matter (Fig. 5C), which corresponds with the distribution of motoneurone cell bodies. Sciatic nerve section did not alter CAT activity in any region of lumbar spinal cord. The activity of CAT in motoneurone cell bodies, therefore, probably was not affected by section of the motoneuron axon.
The effect of dorsal rhizotomy on substance P content, GAD activity and [aHjdiprenorphine binding in the rat dorsal horn In 6 rats with unilateral section of L5 and Le dorsal roots (22-28 days postoperative) the substance P content in the dorsal horn of the operated side was reduced to 20 % of that of the unoperated dorsal horn (Table IA). There was also a 44 ~o decrease in the substance P content of the ventral horn on the operated side while the substance P content of the ventral white matter was unchanged. In the dorsal roots distal to the section there was a 1.87-fold increase in the substance P content compared to the equivalent length of contralateral unoperated dorsal root. In dorsal roots proximal to the section the substance P content was reduced to 23% of the corresponding, unoperated dorsal roots. The substance P content of the dorsal root ganglia and the peripheral part of the sciatic nerve was not significantly changed by dorsal rhizotomy (Table IA). In the lumbar dorsal horn of the same animals there was a 47 ~ decrease in [aH]diprenorphine binding (Table IB) while there was no significant decrease in G A D activity (Table IC).
256 DISCUSSION It has been known for many years that injury to the axon of a neuron results in morphological changes in the neuronal perikarya in addition to Wallerian degeneration in the severed axonal stump (see Lieberman 2s for a review). In primary sensory neurons, which exhibit a pronounced functional and anatomical polarization of their neuronal processes, section of the peripheral axon results in a marked cell loss in spinal 2,~a,as and trigeminal 1,s ganglia which appears to be largely restricted to small neurons 1,~,as. Similar degenerative changes have been reported in the central axon branches and terminals of primary sensory neurons l°,12,2a,24,a2. It is likely, therefore, that the profound depletion of substance P in the dorsal horn and spinal roots following peripheral nerve transection reflects the degeneration of substance Pcontaining primary sensory neurons. In support of this, the time course of substance P depletion appears to parallel the degenerative changes observed in primary afferent terminals13,1s, 23. It is unclear at present whether cell loss occurs exclusively in primary sensory neurons that contain substance P. Some evidence exists for the degeneration of a significant number of larger diameter neurons 1; whether any larger diameter sensory neurons contain substance P remains to be established. There appear to be a number of important differences in the effects of sciatic nerve section and dorsal rhizotomy on the substance P content of the various structural components of primary sensory neurons. Dorsal rhizotomy did not alter the substance P content of the sciatic nerve, which suggests that the transport of substance P from cell bodies to the periphery was unaffected. In contrast, sciatic nerve section produced a depletion of substance P in all parts of the primary sensory neuron, including the cell bodies. These results parallel morphological studies in which chromatolysis and degeneration of dorsal root ganglion cells occurs after peripheral nerve section but not after dorsal rhizotomy 4. In the spinal cord the effect of sciatic nerve section appeared to be confined to the dorsal horn whereas dorsal rhizotomy also produced a significant decrease in substance P in the ventral horn. It is unlikely that the decrease in the substance P content of the ventral horn resulted from non-specific damage since in this case a decrease in the G A D activity in the spinal cord would have been expected. In previous studies Takahashi and Otsuka aa have demonstrated a similar decrease in the substance P content of the cat ventral horn after dorsal rhizotomy. Substance P may, therefore, be contained in some primary afferent fibers which project to the ventral horn. It is also possible that the transsynaptic degeneration of substance P-containing interneurons which originate in the spinal cord could account for the decrease in substance P. H6kfelt et al. 16,17 have described the presence of scattered substance P-positive cell bodies in the spinal cord. It is clear that the origin of substance P in the ventral horn requires a more detailed examination. Morphological studies of synaptic glomeruli in the substantia gelatinosa of the dorsal horn and spinal trigeminal nucleus have shown that the terminals of small diameter afferent fibers receive axoaxonic synapses from two or more classes of substantia gelatinosa interneuronsg,aL The most likely candidates as transmitter
257 substances released by these interneurons are GABA and the opioid peptide enkephalin, both of which are present in a high concentration in dorsal horn interneurons 17,8°,40,al. In the present experiments the decrease in substance P and opiate receptor binding after sciatic nerve section and dorsal rhizotomy provides further evidence that opiate receptors may be located on the terminals of substance Pcontaining primary afferent neurons in the substantia gelatinosa19,26,4L However, the incomplete loss of opiate receptor binding after both operative procedures suggests that only one population of opiate receptors in the dorsal horn are located on primary afferent terminals. G A D activity in the dorsal horn was unchanged by dorsal rhizotomy which is consistent with previous studies showing that the GABA concentration in the dorsal horn is also unchanged 4°,44. In the rat cervical spinal cord, however, a significant decrease in G A D has been reported after dorsal rhizotomyZ2; the reason for the discrepancy between these results is not clear. In the present experiments, the depletion of substance P in the dorsal horn after sciatic nerve section was maintained for up to two months postoperatively. Knyih~ir and Csillik 2z,24 have provided some anatomical evidence that at longer recovery periods after sciatic nerve section, those primary sensory neurons undergoing only partial degeneration may re-establish synaptic contact with second order neurons in the dorsal horn. It would be interesting, therefore, to examine whether substance P levels return to normal with longer recovery periods indicating partial regeneration or possibly the sprouting of surviving neurons. In addition it remains to be established whether neurons in the dorsal horn which respond to both nociceptive stimuli and substance p14,87 exhibit changes in sensitivity to substance P after peripheral nerve injuries, as appears to be the case following dorsal rhizotomy4L ACKNOWLEDGEMENTS Part of this work was supported by research grants from the Japanese Ministry of Education. T.M.J. was supported by a Royal Society Japan Programme Fellowship. We thank Miss H. Taira and Mr. D. Sutoo for excellent technical assistance.
REFERENCES 1 Aldskoglus, H. and Arvidsson, J., Nerve cell degeneration and death in the trigeminal ganglion of the adult rat following peripheral nerve transection, J. Neurocytol., 7 (1978) 229-250. 2 Cavanaugh, M. W., Quantitative effects of the peripheral innervation area on nerves and spinal ganglion cells, J. comp. Neurol., 94 (1951) 181-219. 3 Chan-Palay, V. and Palay, S. L., Ultrastructural identification of substance P cellsand their processes in rat sensory ganglia and their terminals in the spinal cord by immunocytochemistry,Prec. nat. Acad. Sci. (Wash.), 74 (1977) 4050-4054. 4 Cragg, B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 5 Cuello, A. C., Del Fiacco, M. and Paxinos, G., The central and peripheral ends of the substance Pcontaining sensory neurones in the rat trigcminal system, Brain Research, 152 (1978) 499-509. 6 Cuello, A. C., Jessell, T. M., Kanazawa, I. and Iversen, L. L., Substance P: localization in synaptic vesiclesin rat central nervous system, J. Neurochem., 29 (1977) 747-751. 7 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409.
258 8 Galich, J. W., Gregg, J. M. and Duncan, G. H., Quantitative study of trigeminal ganglion response to peripheral nerve trauma, J. Dental Res., 55 (1976) B109. 9 Gobel, S., Golgi studies of the substantia gelatinosa neurons in the spinal trigeminal nucleus, J. comp. Neurol., 162 (1975) 397-416. 10 Gobel, S. and Binck, J. M., Degenerative changes in primary trigeminal axons and in neurons in nucleus caudalis following tooth pulp extirpations in the cat, Brain Research, 132 (1977) 347-354. 11 Goldstein, A., Lowney, L. I. and Pal, B. K., Stereospecific and non-specific interactions of the morphine congener levorphanol in subcellular fractions of mouse brain, Proc. nat. A cad. Sci. (Wash.), 68 (1971) 1742-1747. 12 Goode, G. E., The ultrastructural identification of primary and suprasegrnental afferents in the marginal and gelatinous layers of lumbar spinal cord following central and peripheral lesions, Soc. Neurosci. Abstr., 11, Part 2 (1976) 975. 13 Heimer, L. and Wall, P. D., The dorsal root distribution to the substantia gelatinosa of the rat with a note on the distribution in the cat, Exp. Brain Res., 6 (1968) 89-99. 14 Henry• J. L.• E•ects •f substance P •n functi•na••y identi•ed units in cat spina• c•rd• Brain Resear ch• 114 (1976) 439-451. 15 Htikfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurones, Brain Research, 100 (1975) 235-252. 16 H6kfelt, T., Johansson, O., Kellerth, J. O., Ljundahl, A., Nilsson, G., Nyg~trds,A. and Pernow, B., Immunohistochemical distribution of substance P. In U. S. von Euter and B. Pernow (Eds.), Substance P, Raven Press, New York, 1977, pp. 117-145. 17 H/Skfelt, T. Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemicalanalysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. nat. Acad. Sci. (Wash..), 74 (1977) 3081-3085. 18 Humbertson, A., A chronological study of the degenerative phenomena of dorsal root ganglia cells following section of the sciatic nerve, Anat. Rec., 145 (1963) 244. 19 Jessell, T. M. and Iversen, L. L., Opiate analgesics inhibit substance P release from rat trigeminal nucleus, Nature (Land.), 268 (1977) 549-551. 20 Kanazawa, I., Iversen, L. L. and Kelly, J. S., Glutamate decarboxylase activity in the rat posterior pituitary, pineal gland, dorsal root ganglion and superior cervical ganglion, J. Neurochem., 27 (1976) 1267-1269. 21 Kanazawa, I. and Jessell, T. M., Post-mortem changes and regional distribution of substance P in the rat and mouse nervous system, Brain Research, 117 (1976) 362-367. 22 Kelly, J. S., Gottesfeld, Z. and Schon, F., Reduction in GAD 1 activity from the dorsal lateral region of the deafferented rat spinal cord, Brain Research, 62 (1973) 581-586. 23 Knyihfir, E. and Csillik, B., Effect of peripheral axotomy on the fine structure and histochemistry of the Rolando substance: degenerative atrophy of central processes of pseudounipolar cells, Exp. Brain Res., 26 (1976) 73-87. 24 Knyihfir, E. and Csillik, B., Representation of cutaneous afferents by fluoride-resistant acid phosphatase (FRAP)-active terminals in the rat substantia gelatinosa Rolandi, Acta neurol, scand., 53 (1976) 217-225. 25 Knyihgtr, E., Lfiszl6, I. and Tornyos, S., Fine structure and fluoride-resistant acid phosphatase activity of electron dense sinusoid terminals in the substantia gelatinosa Rolandi of the rat after dorsal root transection, Exp. Brain Res., 19 (1974) 529-544. 26 Lamotte, C., Pert, C. B. and Snyder, S. H., Opiate receptor bindingin primate spinal cord: distribution and changes after dorsal root section, Brain Research, 112 (1976) 407-412. 27 Lembeck, F., Zur Frage der zentralen Ubertragung afferenter Impulse. III. Das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Riickenmarks, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 219 (1953) 197-213. 28 Lieberman, A. R., The axon reaction: a review of the principal features of perikaryal responses to axon injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 29 Lowry, O. H., Rosenbrough, N. J., Farr, A, L. and Randall, R. J., Protein measurement with the Folin phenol reagent. J. biol. Chem., 193 (1951) 265-275. 30 Miyata, Y. and Otsuka, M., Quantitative histochemistry of ~,-aminobutyric acid in cat spinal cord with special reference to presynaptic inhibition,J. Neurochem., 25 (1975) 239-244. 31 Molinoff, P. B. and Kravitz, E. A., The metabolism of T-aminobutyricacid (GABA) in the lobster nervous system - - glutamic decarboxylase, J. Neurochem., 15 (1968) 391-409.
259 32 Moradian, G. P. and Rustioni, A., Transganglionic degeneration in the dorsal horn and dorsal column nucleii of adult rats, Anat. Rec., 187 (1977) 660. 33 Otsuka, M. and Konishi, S., Substance P and excitatory transmitter of primary sensory neurons, Cold Spr. Harb. Syrup. quant. Biol., 40 (1975) 135-143. 34 Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat, Nature (Lond.), 264 (1976) 83-84. 35 Powell, D., Leoman, S. E., Tregear, G. W., Niall, H. D. and Potts, J. T., Jr., Radioimmunoassayfor substance P, Nature New Biol., 241 (1973) 252-254. 36 Pickel, V., Reis, D. J. and Leeman, S. E., Ultrastructural localization of substance P in neurons of rat spinal cord, Brain Research, 122 (1977) 534-540. 37 Randi~, M. and Mileti~, V., Effects of substance P in cat dorsal horn neurons activated by noxious stimuli, Brain Research, 128 (1977) 164-169. 38 Ranson, S. W., Retrograde degeneration in the spinal nerves, J. comp. Neurol., 16 (1906) 265-293. 39 R6thelyi, M. and Szent~tgothai,J., Distributionand connections of afferent fibers in the spinal cord. In A. Iggo (Ed.), Somatosensory Systems, Handbook of Sensory Physiology, Vol. 2, Springer Verlag, Berlin, 1973, pp. 207-270. 40 Roberts, P. J. and Keen, P., Effect of dorsal root section on amino acids of rat spinal cord, Brain Research, 74 (1974) 333-337. 41 Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 2167-2171. 42 Simon, E. J., Hiller, J. M., Crain, S. M. and Peterson, E. R., Demonstration ofstereospecific opiate binding in cultures of fetal mouse dorsal root ganglia and spinal cord, Soc. Neurosci. Abstr., (1977) 301. 43 Takahashi, T., Konishi, S., Powell, D., Leeman, S. E. and Otsuka, M., Identification of the motoneuron-depolarizingpeptide in bovine dorsal root as hypothalamic substance P, Brain Research, 73 (1974) 59-69. 44 Takahashi, T. and Otsuka, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brain Research, 87 (1975) 1-11. 45 Wright, D. M. and Roberts, M. H. T., Supersensitivity to a substance P analogue following dorsal root section, Life Sci., 22 (1978) 19-24.
