Enkephalin and Substance P

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Proc. Nati. Acad. Scf. USA Vol. 74, No. 7, pp. 3081-3085, July 1977 Neurobiology Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalin and substance P (spinal interneurons/descending spinal systems/primary afferents) TOMAS HOKFELT*, AKE LJUNGDAHL*, LARS TERENIUSt, ROBERT ELDE**, AND GORAN NILSSON§ Departments of * Histology and § Pharmacology, Karolinska Institute, Stockholm, and t Department of Medical Pharmacology, Uppsala University, Uppsala, Sweden Communicated by U. S. von Euler, April 25,1977

ABSTRACT The distribution of Met-enkephalin- and sub- in morphine- and stimulation-produced analgesia: the peri- stance P-immunoreactive neurons was studied by indirect im- aqueductal central gray, the medullary raphe nuclei, the spinal munofluorescence in some areas related to pain and analgesia. Met-enkephalin- and substance P-positive cell bodies and nerve trigeminal nucleus, and the spinal cord. It may be significant terminals were observed in the periaqueductal central gray, the that another peptide, substance P, originally discovered by von nucleus raphe magnus, the marginal layer and substantia ge- Euler and Gaddum (23) and structurally characterized by latinosa ofthe spinal trigeminal nucleus, and the dorsal horn Leeman and collaborators (24), is present in the same or adja- of the spinal cord. Lesion experiments suggest that Met-en- cent areas and also in a population of primary sensory neurons kephalin neurons in the dorsal horn and possibly in the spinal (25). In the present paper, the immunohistochemical distri- trigeminal nucleus are interneurons or propriospinal neurons bution of the and substance P systems is compared with nerve terminals in the laminae I and II of tfe cord and in enkephalin the superficial layers of the spinal trigeminal nucleus, respec- with neural substrates involved in pain and analgesia (see refs. tively. These areas are also very rich in substance P-positive 10 and 26). nerve terminals, mainly representing central branches of primary afferent neurons. The present immunohistochemical-anatomical findings MATERIAL AND METHODS support the hypothesis that stimulation-produced analgesia is related to activation of spinal and spinal trigeminal enkephalin Male albino rats (Sprague-Dawley, body weight 150 g) were interneurons forming axo-axonic synapses with (substance P?) divided into three groups: (i) untreated rats, (ii) rats receiving pain afferents in the superficial laminae of the dorsal horn and an injection of 10 or 25 pug of colchicine dissolved in 0.9% the spinal trigeminal nucleus. These interneurons may be acti- (wt/vol) saline (1 gug//l) into the lateral ventricle or the cisterna vated by sensory fibers and by descending fibers from medullary 20 stimulation sites. Transmitter substances in these descending magna, and (iii) rats receiving an intraspinal injection of ,ug fibers may be 5-hydroxytryptamine and substance P. of colchicine dissolved in 0.9% saline (10 ,ug!/ul) at the lumbo- sacral level. The colchicine-treated animals were sacrificed Morphine is a classical agent for the relief of severe, chronic 24-48 hr after the injection. pain. Strong analgesia can also be produced by electric stimu- We have previously observed widespread enkephalin-posi- lation of mesencephalic and medullary structures (refs. 1-9, see tive nerve terminals whereas no immunoreactive cell bodies ref. 10), by acupuncture, or by "electroacupuncture" (11, 12), could be detected (20). Colchicine has been used in other studies a modification of the classical Chinese procedure. Several ob- to increase cell body levels of catecholamines (27), probably due servations suggest a common element in these approaches for to inhibition of axonal transport. pain relief. For instance, narcotic antagonists, such as naloxone Antiserum to Met-enkephalin was raised in rabbits as de- (13), block analgesia induced by electrical stimulation (6-9, 14) scribed elsewhere (20). Crossreaction was about 10% with and by morphine injections (15). Furthermore, tolerance de- Leu-enkephalin and <0.1% with a-, A-, and 'y-endorphins, velops to electrostimulation analgesia and to morphine analgesia substance P, and somatostatin (L. Terenius, unpublished re- and cross tolerance can be observed (16). suIts). Furthermore, because Met-enkephalin occurs in amounts Recently, Hughes and collaborators (17) have identified two 3-4 times greater than those of Leu-enkephalin in rat brain (28), pentapeptides, Leu-enkephalin and Met-enkephalin, that may we ascribe most of the immunofluorescence to Met-enkephalin. represent endogenous ligands for opiate receptors. Available Antiserum against substance P was raised in rabbits as described evidence suggests that the activity profile of endogenous opioids previously (29). is similar to that of the opiates. For instance, enkephalin has The rats were perfused with formalin. After being rinsed, analgesic activity in vivo and the narcotic antagonist naloxone the brain and spinal cord were cut on a cryostat (Dittes, will also counteract enkephalin-induced elevation of pain Heidelberg) and processed for the indirect immunofluorescence thresholds (18, 19). It is therefore conceivable that these ligands technique (see ref. 30). Series of four consecutive sections at may be involved in stimulation-produced analgesia. different levels of the mesencephalon, lower brain stem, and Using the immunohistochemical approach, we have been spinal cord were incubated for 30 min in a humid atmosphere able to outline the gross distribution of enkephalin-positive at 370 with antiserum to Met-enkephalin (diluted 1:10 or 1:20), structures in the central nervous system (refs. 20 and 21, see also substance P (diluted 1:40), and the two control sera, respec- ref. 22). Enkephalin neurons were found in many areas of the tively. The control sera, in which the Met-enkephalin and brain and spinal cord, indicating involvement in many different substance P antibodies were blocked with the respective peptide types of central nervous system functions. Here, however, we (50 jug/ml of serum diluted 1:10), were included to establish focus attention strictly on areas assumed to be of importance the specificity of the staining. After a rinse in phosphate-buffered saline, the sections were incubated with fluorescein iso- * Present address: Department of Anatomy, University of Minnesota, thiocyanate-conjugated antibodies (SBL, Stockholm; diluted Minneapolis, MN. 1:4) under the same conditions as described above, rinsed in 3081 Downloaded by guest on September 29, 2021 3082 Neurobiology: Hokfelt et al. Proc. Natl. Acad. Sci. USA 74 (1977)

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FIG. 1. Immunofluorescence studies. Arrowhead always points dorsally. DF, dorsal funiculus; P, tractus corticospinalis; asterisk indicates central canal. (Bar indicates 50Mm.) Anatomic sites: dorsal horn ofthe spinal cord (A, B); nucleus raphe magnus (C); intermediate gray matter of the sacral spinal cord (D-F). Treatments: untreated (A, B); colchicine-treated (C-F). Sera: antiserum to substance P (A, C, E); antiserum to Met-enkephalin (B, D); enkephalin control serum (F). A and B and D, E, and F are consecutive sections, respectively. A high density ofsubstance P-positive nerve terminals was seen in laminae I and II of the dorsal horn (A). The rectangle in A indicates approximately the area shown in B. The enkephalin-positive nerve terminals had a parallel but less dense distribution as seen at the higher magnification (B). Note lack of enkephalin-positive fibers in the zone of Lissauer (arrow in B) whereas numerous substance P-positive fibers are present in this area (arrow in A). Numerous substance P-immunoreactive cell bodies were seen in the nucleus raphe magnus (C) after colchicine treatment. At the sacral level both enkephalin- (arrows in D) and substance P-positive (arrows in E) cell bodies were seen after intraspinal colchicine injections. Note the networks ofenkephalin- (D) and substance P-positive (E) nerve terminals. No fluorescent neurons or nerve terminals were seen after incubation with control serum (F).

phosphate-buffered saline, mounted in phosphate-buffered RESULTS saline/glycerol, 1:3 (vol/vol), and examined in a Zeiss fluores- In untreated rats, extensive networks of Met-enkephalin- and cence microscope. The anatomical nomenclature is according substance P-positive nerve terminals were observed in the to Palkovits and Jacobowitz (31). periaqueductal central gray and in the marginal layer and Downloaded by guest on September 29, 2021 Neurobiology: Hakfelt et al. Proc. Natl. Acad. Sci. USA 74 (1977) 3083 I

FIG. 2. Immunofluorescence studies of the dorsal horn of the spinal cord (lower lumbar and upper sacral level) of colchicine-treated rat after incubation with antiserum to Met-enkephalin. DF, dorsal funiculus. (Bar indicates 50 Am.) Cell bodies (arrows) are observed in superficial (B) and deeper laminae (C). B and C represent higher magnifications ofA as indicated by rectangles. Note that the fluorescent nerve terminals are less bright compared to Fig. 1B. substantia gelatinosa of the spinal trigeminal nucleus. It is im- observed. The results described above are summarized in Fig. portant to note that both enkephalin- and substance P-positive 3. fibers were present only in the caudal parts of the spinal tri- Met-enkephalin- and substance P-positive cell bodies, axons, geminal nucleus and that there was a striking overlap between and nerve terminals were observed also in many other areas of the distribution patterns of the nerve terminals containing these the central nervous system. A full account of the localization two peptides. In the medullary raphe nuclei (nucleus raphe of these neurons will be given elsewhere. magnus and nucleus raphe pallidus), low to moderate concen- None of the immunoreactive structures described above were trations of substance P-positive and Met-enkephalin-positive observed after incubation with control sera (Fig. IF). nerve terminals were observed. In the spinal cord, the highest concentrations were observed in laminae I and 11 (32) (Fig. 1 A and B) with moderately dense networks in laminae IV-VII, DISCUSSION the ventral horn, and the area around the central canal. In the There is evidence that morphine and electrical stimulations of first two laminae, there were considerably more substance some brain loci and of peripheral nerves produce analgesia via SP-positive than Met-enkephalin-positive fibers. Whereas the the same type of receptors and that the enkephalins represent zone of Lissauer contained numerous cross-sectioned substance important endogenous ligands for these receptors. The present P-positive fibers, no immunoreaction with Met-enkephalin findings demonstrate enkephalin-positive nerve terminals in antiserum could be observed. No Met-enkephalin- or substance all areas where morphine is known to produce behavioral an- P-immunoreactive cell bodies could be observed in any of these algesia, including the periaqueductal gray, the medullary raphe areas. 4 nuclei, the caudal trigeminal nucleus, and the spinal cord. It In colchicine-treated rats, numerous Met-enkephalin- and has been inferred from several studies that the ultimate site of substance P-positive cells were seen in addition to nerve ter- action of midbrain stimulation would in fact be the spinal tri- minals. Generally, the immunofluorescence of nerve terminals, geminal nucleus or the spinal cord (refs. 5 and 33-36, see ref. especially the enkephalin-positive ones, appeared less strong 10). It is possible that, in electroacupuncture, activation of en- and distinct in colchicine-treated rats compared to untreated dorphin systems also occurs at the segmental level because this rats. increases spinal cerebrospinal fluid levels of endorphins (37). Met-enkephalin- and substance P-immunoreactive cell bodies Our findings indicate that at the spinal level the enkephalin were observed in the ventrolateral parts of the periaqueductal system is either interneuronal or propriospinal. Both en- central gray and in the raphe magnus (Fig. IC) and raphe kephalin-positive terminals and cell bodies were found in pallidus nuclei. In the spinal trigeminal nucleus, many Met- the dorsal horn. Neither transection of the cord at the thoracic enkephalin-positive cell bodies were observed in the marginal level nor unilateral dorsal rhizotomy at the lumbar and sacral layer of its caudal part whereas substance P-positive cells were level produced any marked changes in the concentration of observed only occasionally. Numerous Met-enkephalin- and enkephalin-positive nerve terminals in the lumbar cord (38). substance P-immunoreactive cell bodies were observed in the The presence of enkephalin nerve terminals and enkephalin spinal cord (Figs. 1 D and E and 2). They were present mainly cell bodies in the marginal layers and substantia gelatinosa of in laminae I-V. There were more Met-enkephalin-positive than the spinal trigeminal nucleus may indicate the existence of substance P-positive cells in the dorsal horn of the spinal cord. enkephalin interneurons also in this nucleus. It is suggested that Strongly fluorescent substance P-positive axons were present activation of enkephalin interneurons in the spinal trigeminal in the caudal medulla oblongata ventromedially to the decus- nucleus and in the dorsal horn is the last and critical step in satio pyramidis. In this area, substance P-positive cells were also stimulation-produced analgesia. This finding supports con- Downloaded by guest on September 29, 2021 3084 Neurobiology: Hbkfelt et al. Proc. Nati. Acad. Sci. USA 74 (1977) iate receptors in primary sensory afferents Enkephalin neurons may therefore form axo-axonic synapses (see refs. 26 and 40) on primary afferents, possibly on substance P afferents. The remarkable overlap of enkephalin and substance P nerve ter- minal fields in the superficial layers of the spinal trigeminal nucleus and in laminae I and II of the dorsal horn is striking. Which central pathways may be involved in the activation of spinal enkephalinergic neurons? Both the periaqueductal central gray and medullary raphe nuclei are effective stimulation sites for producing analgesia, suggesting that descending systems (41) must be involved. From the periaqueductal central gray, descending projections to the spinal cord have been described in the cat (42) but so far not in the rat. From the medullary raphe nucleus of the rat, on the other hand, Brodal et al. (43) have demonstrated direct projections to the cord. In a combined neuroanatomical and electrophysiological study, Basbaum et al. (35, 36) analyzed the descending system involved in stimulation-produced analgesia (see also ref. 13). The cell bodies are located in the nucleus raphe magnus and the axons appear to run in the dorsolateral part of the lateral funiculus, terminating in the dorsal horn. Basbaum et al. (35, 36) suggested, on the basis of preliminary experimental evidence, that there is a circuitry from the periaqueductal central gray to the spinal cord via the nucleus raphe magnus. With regard to the neurohumoral substrate of the medullary descending system, both morphological evidence (44) and pharmacological analysis (45, 46) suggest that a 5-hydroxytryptamine projection may represent one link between the stimulation site and the spinal cord. The present study dem- onstrates both enkephalin- and substance P-positive cell bodies in these nuclei. Whereas no evidence for descending enkephalin neurons exist, descending substance P pathways have in fact been demonstrated (38) and it may be that substance P pathways represent part of the system related to stimulation-produced analgesia described by Basbaum et al. (35, 36). Because substance P appears to exert an excitatory action (47), it could activate the enkephalin interneurons of the dorsal horn. In- terestingly, intraventricular or systemic administration of substance P has in fact been found to produce naloxone-re- versible analgesia (48, 49). 5-Hydroxytryptamine, on the other hand, appears mainly to be inhibitory and the analgesic effect D . mediated by descending 5-hydroxytryptamine systems has been FIG. 3. Distribution of enkephalin-positive (left side) and sub- ascribed to an axo-axonic influence on primary afferent nerve stance P-positive (right side) cell bodies (asterisks) and nerve ter- terminals in the dorsal horn (50). minals (dots) at three levels of the lower brain stem (A = P 0.5 mm; Primary afferent neurons may also be involved in activation B = P 4.5 mm; C = P 7.4 mm) and spinal cord. Note that enkephalin- of enkephalin interneurons. However, the type of primary af- and substance P-positive cell bodies and nerve terminals are present in many more areas than indicated on this drawing. AF, anterior ferent neurons activated in classical acupuncture or electro- funiculus; cod, nucleus cochlearis dorsalis; DF, dorsal funiculus; FLM, acupuncture is not known, and we can only speculate about fasciculus longitudinalis medialis; gr, nucleus gracilis; io, nucleus ol- the primary afferent fibers that might activate the enkephalin ivaris inferior; LF, lateral funiculus; LL, lemniscus lateralis; LM, neurons. There is now strong biochemical (51), neurophysio- lemniscus medialis; nrv, nucleus reticularis medullae oblongatae pars logical (47), and immunohistochemical (25) evidence that ventralis; nts, nucleus tractus solitarii; ntV, nucleus tractus spinalis substance P is present in some primary sensory neurons. Other nervi trigemini (spinal trigeminal nucleus); ntVd, nucleus tractus primary afferent neurons may contain a somatostatin-like spinalis nervi trigemini pars dorsomedialis; nVII, nucleus originis nervi facialis; P, tractus corticospinalis; PCI, pedunculus cerebellaris peptide (52). The substance P neurons have small cell bodies inferior; PCS, pedunculus cerebellaris superior; rd, nucleus raphe and fine-caliber axons with peripheral branches in most tissues. dorsalis; rgi, nucleus reticularis gigantocellularis; rl, nucleus reticularis Their central branches terminate mainly in laminae I and II lateralis; rm, nucleus raphe magnus; rp, nucleus raphe pallidus; SGC, but probably also in deeper laminae of the dorsal horn. This substantia grisea centralis; TSV, tractus spinalis nervi trigemini; vm, distribution and these morphological characteristics are inter- nucleus vestibularis medialis. The nomenclature as well as the cross in of the results of Christensen and Perl (53) who sections of the lower brain stem are according to Palkovits and Ja- esting view cobowitz (31). found that cells responding to noxious stimuli are located mainly in lamina I. Furthermore, they may be considered to add fur- clusions drawn by LaMotte et al. (39). They demonstrated high ther support for the view expressed by Henry (54) that sub- opiate receptor binding in the superficial laminae of the dor- stance P "may have an excitatory role in central transmission sal horn and a marked decrease in the binding after dorsal in spinal pain pathways acting ... possibly at the first afferent rhizotomy. They suggested presynaptic localization of the op- synapse." Downloaded by guest on September 29, 2021 Neurobiology: H6kfelt et al. Proc. Natl. Acad. Sci. USA 74 (1977) 3085

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