Opioid Peptide Lmmunoreactivity in Spinal and Trigeminal Dorsal Horn Neurons Projecting to the Parabrachial Nucleus in the Rat

The Journal of Neuroscience May 1986, 135): 1220-1226

Opioid Peptide lmmunoreactivity in Spinal and Trigeminal Dorsal Horn Neurons Projecting to the Parabrachial Nucleus in the Rat

David G. Standaert,* Stanley J. Watson,t Richard A. Houghten,$ and Clifford B. Saper** “Departments of Neurology and Neurological Surgery (Neurology) and Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, TMental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, *Scripps Clinic, Department of Molecular Biology, La Jolla, California 92137, and **Departments of Pharmacological and Physiological Sciences and Neurology, and Brain Research Institute, University of Chicago, Illinois 60637

The parabrachial nucleus (PB) is the major relay for ascending demonstrated projections from the spinal cord to PB in several visceral afferent information from the nucleus of the solitary species (Mehler, 1962; Mehler et al. 1960; Zemlan et al., 1978). tract to the forebrain. We have recently found that PB in the The spinoparabrachial projection has recently been examined rat also receives a substantial afferent projection from neurons in detail in the rat and the cat using the anterograde and retro- in the marginal zone of the entire length of the spinal and tri- grade transport of wheat germ agglutinin conjugated to horse- geminal dorsal horn. Immunoreactive perikarya stained with radish peroxidase (WGA-HRP) (Cechetto et al., 1985; Panneton antisera against several neuropeptides -including dynorphin, and Burton, 1985). Injections of WGA-HRP into PB retro- efikephalins, and substance P-have been identified in the gradely label large, flattened cells that lie parallel to the pial marginal zone. We therefore investigated the chemical specific- surface within the marginal zone of the dorsal horn, throughout ity of the spinoparabrachial projection by combining fluorescent the entire rostrocaudal extent of the spinal cord and spinal tri- retrograde tracing with immunofluorescence for substance P, geminal nucleus pars caudalis. At the level of the obex, an ad- dynorphin Al -17, met-enkephalin, and two enkephalin precur- ditional group of retrogradely labeled neurons is seen among sor fragments (proenkephalin 192-203 and peptide E). the fibers of the dorsal part of the spinal trigeminal tract in the Following PB injections of fluorescent dyes, about half of the promontoriuml of the spinal trigeminal nucleus. Following in- retrogradely labeled neurons in the marginal zone stained with jection of WGA-HRP into the marginal zone of the spinal cord, antisera against either dynorphin or enkephalin series peptides. anterogradely labeled fibers can be seen innervating predomi- Elution-restaining experiments indicated that the dynorphin- nantly the lateral division of PB, including the central, dorsal, and enkephalin-immunoreactivities were contained within sep- and external lateral subnuclei and the Kolliker-Fuse nucleus. arate populations of marginal zone neurons. We could not iden- Injections of WGA-HRP into the promontorium and adjacent tify any substance P-immunoreactive perikarya in the marginal spinal trigeminal nucleus label fibers innervating these same zone, but substance P-immunoreactive fibers were seen in close subnuclei of lateral PB, as well as the medial subdivision of PB apposition to retrogradely labeled, opioid-immunoreactive cell (Cechetto et al., 1985; Fulwiler and Saper, 1984; Panneton and bodies and dendrites. Burton, 1985). These results indicate that the dynorphin- and enkephalin- Neurons in the outer lamina of the dorsal horn have been immunoreactive perikarya in the marginal zone of the dorsal observed to stain immunohistochemically for a number of neu- horn represent independent neuronal populations. These opioid- ropeptides, including enkephalins (Aronin et al., 198 1; Glazer immunoreactive neurons, which are believed to have extensive and Basbaum, 198 I), dynorphin (Khachaturian et al., 1982), local collateral connections, are the main source of a long as- and substance P (Del Fiacco and Cuello, 1980; DiFiglia et al., cending projection to the parabrachial nucleus in the rat. Fur- 1982; Ljungdahl et al., 1978). Immunohistochemical staining thermore, opioid neurons in the marginal zone may receive sub- for all three of these peptides has been observed in fibers forming stance P-immunoreactive primary sensory afferents. dense terminal fields in PB (Kawai et al., 1982; Khachaturian et al., 1982; Uger et al., 1983; Milner et al., 1984). Each of The parabrachial nucleus (PB) is an important relay for visceral these peptides is therefore a candidate for the transmitter of the afferent information traveling from the nucleus of the solitary spinoparabrachial pathway. tract to the forebrain. Early studies of ascending spinal cord In the present study, we have used a fluorescent retrograde pathways using anterograde degeneration after spinal lesions neuronal tracer in combination with immunohistochemistry to investigate the peptides present in spinal and trigeminal neurons projecting to PB. Our results demonstrate that most of the neu- Received Jan. 29, 1985; revised Oct. 15, 1985; accepted Oct. 15, 1985. rons in the spinal and trigeminal dorsal horns that project to We thank Ms. Quan Hue Ha for technical assistance and Ms. Patti Nacci for PB contain either dynorphin- or enkephalin-like immunoreac- typing the manuscript. The met-enkephalin antiserum was the generous gift of tive (-lir) material. Dr. R. Elde. Supported by grants from USPHS NS18669 and N522835 (C.B.S.), NS18I 13 (R.A.H.), and GM07200 (D,G.S.); from ADAMHA DA 02265 and MH 397 17 (S.J.W.); and from the T, Raphael Research Fund (S.J.W.). C.B.S. is recipient of NINCDS Teacher-Investigator Development Award NS0063 1 and a M&night Scholar Award, I Although the term “paratrigeminal nucleus” has also been used for this cell Correspondence should h addressed to Dr. Saper, Department of Pharmacolog- group (Chan-Palay, 1978; Panneton and Burton, 1985), Rhoton et al. (1966) earlier ical and Physiological Sciences, University of Chicago, 947 East 58 th Street, Chicago, used this term to describe a group of cells lateral to the spinal trigeminal tract at IL 60637. the pontomedullary junction in the monkey. Because of this ambiguity, we prefer Copyright 0 1986 Society for Neuroscience 0270-6474/86/05 1220-07$02.00/O Astrom’s (1953) term, “promontorium.”

1220 The Journal of Neuroscience Opioid Peptide lmmunoreactivity in Rat 1221

Materials and Methods preincubating the diluted serum with synthetic dynorphin A l-l 7 (Bach- em), leu-enkephalin (Sigma), met-enkephalin (Sigma), or the proen- Retrograde transport-immunohistochemistry experiments kephalin 192-203 fragment at a concentration of 50 pg peptide/ml of Stereotaxic injections of 50 nl of a 5% solution of Fast Blue dye (Illing) diluted serum. were made into the PB region of six Sprague-Dawley rats. Injections The specificity of the substance P antibody was tested by adsorption were made using a glass micropipette syringe (Saper, 1983) under chloral with 50 rg of synthetic substance P (Sigma) per milliliter of diluted hydrate anesthesia. After 7-14 d survival, the rats were treated with antibody. 150 pg colchicine (Sigma) in 10 ~1 saline injected stereotaxically into the lateral ventricle. Between 24 and 48 hr later, the rats were reanesthe- tized with chloral hydrate and perfused through the heart with saline Results followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 The brains and spinal cords were removed, and the spinal cords were Retrograde transport-immunohistochemistry experiments cut into blocks of three segments each. The brain stems were cut into In all six rats, large depositsof Fast Blue dye were seenin the 50 pm sections using a freezing microtome. Twelve 50 pm sections were PB region. The injection sites included both the medial and cut from the rostra1 end of each spinal cord block. The pontine sections lateral PB subnuclei, as well as the Kolliker-Fuse nucleus, the were mounted on slides and used to verify the injection site. The med- locus ceruleus, and the mesencephalicand principal sensory ullary and spinal cord sections were processed for immunohistochem- trigeminal nuclei. Retrogradely labeled cell bodies were ob- istry. served in the promontorium, as well as throughout lamina I of Sections were incubated for 1 hr in 0.1 M phosphate buffer, pH 7.3- 0.9% NaCl (PBS), containing 0.25% Triton X-160, followed by 1 hr in the spinal and trigeminal dorsal horns, bilaterally. As described PBS containing 3% goat serum (PBS-G). The sections were then divided in our previous study, this pattern of retrograde labelingis pro- into four groups of every fourth section. Each group of sections was duced only by injections involving PB, and not by injections incubated overnight at 4°C in PBS-G containing both substance P an- into adjacent structures (Cechetto et al., 1985). tibody diluted 1:2000 or 1:4000, and one of the following sera: anti- Cellscontaining dynorphin-, met-enkephalin-,peptide E-, and dynorphin A l-l 7 ( 1: 1000), anti-peptide E ( 1:300), anti-proenkephalin proenkephalin-lir were found in the trigeminal promontorium 192-203 (1:300), or anti-met-enkephalin (1:200). The next day, the and throughout the marginal zone of the spinal and trigeminal sections were incubated for 1 hr in fluoroscein isothiocvanate (FITC)- dorsal horn. All four of the antisera stainedpopulations of neu- labeled goat anti-rabbit serum (Miles; 1:50 in goat serum-PBS), washed rons that were similar in appearanceand distribution to the cells several times in PBS-G, and incubated for an additional hour in rho- of origin of the spinoparabrachialprojection. Many of the opioid damine isothiocyanate (RITC)-labeled rabbit anti-mouse serum (Miles; I:50 in PBS-G). The sections were then mounted on gelatin-coated slides cells were also labeledwith the retrograde tracer. Becauseof the and coverslipped with glycerin. light immunohistochemical staining of someof the cell bodies, and heavy staining of many fibers in the sameregion, it was not Antibody elution experiments possibleto determine exactly what proportion of the cells containing the retrograde tracer were immunohistochemically la- The elution-restaining technique of Tramu et al. (1978) was used to beled. However, in some casesas many as half of the cells in stain the same sections sequentially for two different opioid peptides. In all, six animals were used for these experiments. All were treated the marginal zone and promontorium that contained Fast Blue with 150 pg of colchicine and perfused as described above. Sections also stainedwith either dynorphin, met-enkephalin,proenkeph- from the medulla and spinal cord were incubated overnight in PBS-G alin, or peptide E antisera (Figs. 1, 2). containing substance P antibody (1:4000) and either met-enkephalin or In the spinal cord, control incubations usingmet-enkephalin dynorphin A 1- 17 antisera as described above. The next day, these sec- and dynorphin antisera preadsorbedwith synthetic dynorphin tions were incubated in PBS-G containing FITC-labeled goat anti-rabbit or met-enkephalin showed that each peptide blocked the ap- (Miles. 1:50) and RITC-labeled aoat anti-rat (Cannel. 1:50) antisera for propriate antiserum and did not affect the other. Neither anti- 1 hr. The sections were then coverslipped with a-solution of PBS and serumwas affected by synthetic leu-enkephalin.The proenkeph- glycerin (1:3). After observing and photographing immunohistochemically stained alin stainingwas not affected by dynorphin, met-enkephalin,or neurons in the promontorium and dorsal horn, the coverslips were leu-enkephalin, but was completely blocked by proenkephalin removed and the sections washed in PBS. Antibodies were eluted using 192-203. The peptide E stainingwas blocked by dynorphin and a solution of 0.1% KMnO, and 0.20% HSO, in distilled water for 5- met- and leu-enkephalin. 10 min. After decolorizing in 0.5% NaH;Sd, for 1 min, the sections SubstanceP-lir fibers were found to innervate the promon- were incubated in FITC-labeled goat anti-rabbit serum for 30 min, torium and the outer lamina of the spinal trigeminal nucleus coverslipped with PBS-glycerin, and observed to ascertain that all stain- and spinal dorsal horn, as has been reported by other investi- ing had been eliminated. The coverslips were removed, and the sections gators (Barber et al., 1979; Del Fiacco and Cuello, 1980). No were then washed in PBS and incubated overnight in a second opioid unequivocal substanceP-lir cell bodies were observed within primary antiserum (i.e., sections that had been stained with anti-met- enkephalin were incubated in anti-dynorphin, while sections stained the dorsal horn, although the intensefiber stainingthat persisted with anti-dynorphin were incubated with anti-met-enkephalin). The despite colchicine treatment might have made cell bodies dif- next day, the sections were incubated for 1 hr in FITC-labeled goat anti- ficult to distinguish. A notable feature of the substanceP-stain- rabbit serum. The sections were coverslipped with PBS-glycerine, and ing was the relationship between the substanceP-lir fibers and the new pattern of staining in the promontorium was compared with opioid-lir cells. SubstanceP-lir fibers were observed surround- the photographs of the staining produced by the first antiserum. ing and outlining individual cell bodies stainedwith dynorphin, met-enkephalin,proenkephalin, or peptide E antisera; many of Antisera thesealso contained the retrograde tracer (Fig. 1, C and D). This The methionine-enkephalin antiserum was obtained from Dr. R. Elde relationship was most conspicuousin the promontorium, while (#R153e); the production and specificity of this serum have been de- in the spinal cord the substanceP-lir fiber stainingwas so intense scribed (Haber and Elde, 1982). The rat monoclonal substance P an- that it was only rarely possibleto discerna relationship between tibodv was obtained from Sera-lab (MAS 035). The prenaration and individual fibers and cells. specihcity of this antibody have been described’by Cuellokt al. (1979). All substanceP immunohistochemicalstaining was abolished Antisera to dynorphin Al-17 (#54-B16), proenkephalin 192-203 (#52- B15), and peptide E (#63-B16) were prepared (by S.J.W.) using antigens by pre-incubating the antibody with synthetic substanceP. The made by conjugating peptide E, dynorphin Al-17, or bovine proen- patterns of staining demonstrated using RITC-labeled anti-rat kephalin 192-203 (prepared by R.A.H.) to thyroglobulin using glutar- IgG produced in rabbit (retrograde transport experiments)or in aldehyde. goat (elution experiments)were identical. Using either method, Specificity of the opioid antisera in our preparations was tested by substanceP-lir fiberswere found in closeassociation with opioid- Standaert et al. Vol. 6, No. 5, May 1966

Figure 1. Fluorescence photomicrographs of neurons in the dorsal horn of the spinal cord. A, Low-power view of retrograde label in dorsal horn at C8 level. Arrow indicates cell shown at higher magnification in RD. S, Fast Blue retrograde label; C, dynorphin-like immunoreactivity(-lir); D, substance P-lir. E, Retrograde label in the dorsal horn at T5 level. F-H, Same cell at higher magnification; F, Fast Blue; G, peptide E-liq H, substance P-hr. Note the punctate appearance of substance P-lir staining around these two cells. Scale bar: A and E, 100 pm; B-D and F-H, 10 flrn. The Journal of Neuroscience Opioid Peptide lmmunoreactivity in Rat 1223

Figure 2. Fluorescence photomicrographs of neurons in two sections through the promontorium of the spinal trigeminal tract. A and E, LOW- power view of Fast Blue retrograde labeling shown at higher power in B and F, respectively; C, proenkephalin-lir; G, met-enkephalin-lir; D and H, substance P-hr. Note that the retrogradely labeled dendrite (arrow) in B stained immunohistochemically for proenkephalin 192-203 (C). This process was also invested with substance P-lir terminals (0). By focusing through the section, it was apparent that this cell body was continuous with the proenkephalin-lir retrogradely labeled cell body (arrowhead) in C. Note that a few substance P-lir terminals were also visualized adjacent to the met-enkephalin-lir, retrogradely labeled neuron shown in F and G. Scale, as in Figure 1. 1224 Standaert et al. Vol. 6, No. 5, May 1986

Figure 3. Photomicrographsof a sectionthrough the promontoriumstained sequentially for enkephalin-and dynorphin-hr.A, Low-power view of sectionstained for met-enkephalin-lir. B, Same section at higher magnification. Solid arrows identify three enkephalin-lir neurons. C, Same section,after elutionof antibodies,stained for dynorphin-lir.D, Highermagnification showing that the threeenkephalin-lir neurons (solid arrows) are not stainedby dynorphinantiserum. Open arrow identifiesa dynorphin-lir neuronthat is not stainedfor enkephalin-lir.Scale bar: A and C, 125 pm; B and D, 50 pm.

staining neurons. Neither method revealed any single structure the dye may be picked up by fibers of passage.As a result, in stained simultaneously for substanceP and one of the opioids. studies using this tracer alone, it is not possible to determine These observationsdemonstrate that both of thesemethods are precisely where the axons of retrogradely labeled neurons ter- capableof simultaneousindependent localization of two differ- minate. Fortunately, in our earlier study on the spinoparabra- ent peptides. chial projection, we establishedthat none of the structures adjacent to PB that might have been included in the Fast Blue E&ion experiments injection sitesreceive projections from the marginal zone of the As both the dynorphin and enkephalin family antisera stained dorsal horn. The one spinal pathway that passesthrough this similar-appearingpopulations of lamina I dorsal horn neurons, region, the ventral spinocerebellar tract, does not arise from we attempted to determine whether the antiserawere staining neuronsin the marginal zone of the dorsal horn in the rat (Ma- the samecells. Using the elution technique ofTramu et al. (1978) tsushita and Hosoya, 1979; Snyder et al., 1978). Furthermore, as described, we were able to stain sections sequentially for the retrogradely labeled neurons observed in this study were enkephalin and dynorphin. We found a large number of pro- essentially identical both in number and distribution to the montorium and dorsal horn cells that stained for each of these spinoparabrachialneurons described in our earlier study using two peptides, but no examples were seenof cells that stained WGA-HRP, a tracer that is not picked up by fibers of passage for both antisera (Fig. 3). (Brodal et al., 1983; Shook et al., 1984). We therefore conclude that the retrogradely labeled neuronsin the promontorium and Discussion in lamina I of the spinal and trigeminal dorsal horn in these In a previous report we described a pathway that arisesfrom experiments actually innervate PB. neuronsin the marginalzone of the spinal and trigeminal dorsal The antisera we used in this study appear to stain separate horns and terminates in PB. The present experiments demon- populations of enkephalin and dynorphin neurons in the para- strate that the cells of origin of this pathway can be stained ventricular nucleus of the hypothalamus (Watson et al., 1982). immunohistochemically using antisera to peptides in the dy- The resultsof our elution experiments suggestthat the sameis norphin and enkephalin families. Furthermore, we have pre- true of neurons in the promontorium and dorsal horn. This sentedevidence suggestingthat enkephalin- and dynorphin-lir finding is in agreement with the recent report of Guthrie and are present within separatepopulations of neurons. Basbaum (1984; and personal communication). In individual The retrograde tracer usedin this study, Fast Blue, has two retrograde tracing experiments, we found that as many as half inherent disadvantages:the injection sitesare quite large, and of the retrogradely labeled promontorium and dorsal horn neu- The Journal of Neuroscience Opioid Peptide lmmunoreactivity in Rat 1225 rons stained for each of the opioid peptides. If these peptides trigeminal, facial, glossopharyngeal, and vagal nerves and their nuclei are, in fact, contained in separatepopulations of neurons,then in the mouse. Acta Phvsiol. Stand. (SUDD~. 6) 29: 209-320. most, if not all, of the spinoparabrachialneurons must contain Barber, R. P., J. E. Vaughn, J. R. Slemmon; P. M. Salvaterra, E. Roberts, and S. E. Leeman (1979) The origin, distribution, and synaptic opioid peptides. relationships of substance P axons in rat spinal cord. J. Comp. Neurol. We have observed that many of the opioid-lir neuronsof the 184: 331-352. dorsalhorn that project to PB are surroundedby densenetworks Bennett, G., M. Abdelmoumene, H. Hayashi, and R. Dubner (1980) of substanceP-lir fibers. While it is not possible to determine Physiology and morphology of substantia gelatinosa neurons intra- by light microscopy whether synaptic contacts are made, the cellularly stained with horseradish peroxidase. J. Comp. Neurol. 194: pattern of varicosities observed surrounding the opioid neurons 809-827. is highly suggestiveof interaction between thesefibers and the Brodal, P., E. Dietrichs, J. G. Bjaalie, T. Nordby, and F. Walberg (1983) opioid cells. Previous investigators have commented on the Is lectin-coupled horseradish peroxidase taken up and transported by close associationof substanceP-lir fibers and enkephalin-lir undamaged as well as by damaged fibers in the central nervous system? Brain Res. 278: l-9. neuronsin the spinal dorsal horn. However, it has proved quite Brown, A. G. (1982) Dorsal horn of the spinal cord. Q. J. Exp. Physiol. difficult to demonstrate synaptic contact between these ele- 67: 193-212. ments, leading to the hypothesisof “nonsynaptic” interactions Cechetto, D. F., D. G. Standaert, and C. B. Saper (1985) Spinal and (Cuello, 1983). trigeminal dorsal horn projections to the parabrachial nucleus in the The antibody used to stain these fibers was raised against rat. J. Comp. Neurol. 240: 153-160. substanceP, but it is important to note that substanceP is only Chan-Palay, V. (1978) The paratrigeminal nucleus: I. Neurons and one of a family of neuropeptides, the tachykinins, that also synaptic organization. J. Neurocytol. 7: 405-4 18. includes substanceK and neuromedin K. These peptides all Cuello, A. C. (1983) Central distribution of opioid peptides. Br. Med. sharea common C-terminal sequence,and all three are thought Bull. 39: 11-16. Cuello, A. C., G. Galfre, and C. Milstein (1979) Detection of substance to be presentin mammalian CNS (Harmar, 1984).The mono- P in the central nervous system by a monoclonal antibody. Proc. Natl. clonal substanceP antibody usedin this investigation probably Acad. Sci. USA 76: 3532-3536. cross-reactswith all of thesepeptides (J. Maggie, personalcom- Del Fiacco, M., and A. C. Cue110 (1980) Substance P- and enkephalin- munication). Therefore, although we refer to the staining as containing neurons in the rat trigeminal system. Neuroscience 5: 803- substanceP-lir, the actual peptide contained in thesefibers re- 815. mains unknown. DiFiglia, M., N. Aronin, and S. E. Leeman (1982) Light microscopic Most of the enkephalin-lir neurons in the dorsal horn are and ultrastructural localization of substance P in the dorsal horn of thought to be interneurons, and there is considerableevidence the monkey spinal cord. Neuroscience 7: 1127-l 139. that opioid peptidescan regulatethe releaseof substanceP from Fulwiler, C. E., and C. B. Saper (1984) Subnuclear organization of the efferent connections of the uarabrachial nucleus in the rat. Brain Res. primary sensory afferents (seeJesse& 1983, for review). This Rev. 7: 229-259. interaction hasbeen interpreted as a mechanismfor modulating Glazer, E., and A. Basbaum (198 1) Immunohistochemical localization the transmissionof nociceptive information. Our results indi- of leu-enkephalin in the spinal cord of the cat: Enkeohalin-containine cate that many of the neuronsin the dorsal horn that stain for marginal neurons and pain modulation. J. Comp. Neural. 196: 3771 opioid peptideshave long ascendingprojections to PB. Whether 389. enkephalin-lir terminals in the dorsal horn are collaterals of Guthrie, J., and A. Basbaum (1984) Colocalization of immunoreactive spinoparabrachialaxons or arise from a secondpopulation of proenkephalin and prodynorphin products in medullary neurons of enkephalin-lir interneurons remains to be determined. How- the rat. Neuropeptides 4: 437-445. ever, many of the large, flattened lamina I neuronsthat receive Haber, S., and R. Elde (1982) The distribution of enkephalin immunoreactive terminals in the monkey central nervous system: An nociceptive afferents have both local recurrent and long as- immunohistochemical study. Neuroscience 7: 1049-1095: cending axon collaterals (Bennett et al., 1980; Brown, 1982; Hamilton, R. B.. H. Ellenberaer. D. Liskowskv. and N. Schneiderman Light et al., 1979;Woolf and Fitzgerald, 1983).Opioid neurons (198 1) ‘Parabrachial area as mediator of bradycardia in rabbits. J. in the dorsal horn may therefore be involved both in local Autonom.Nerv. System4: 26l-28 1. transmissionof nociceptive information, as well asin regulating Harmar,A. J. (1984) Three tachykinins in mammalian brain. Trends autonomic responsesto painful stimuli by meansof their input Neurosci. 7: 57-60. to PB. Jansc6, G., and E. Kiraly (1980) Distribution of chemosensitive pri- An alternate hypothesisis suggestedby the observation that mary sensory afferents in the central nervous system of the rat. J. neonatal capsaicintreatment eliminates substanceP-lir fibers Comp. Neurol. 190: 78 l-792. in the dorsal horn and promontorium. This has been taken to Jessel, T. (1983) Nociception. In Bruin Peptides, D. T. Krieger, M. J. Brownstein, and J. B. Martin, eds., pp. 315-332, Wiley-Interscience, imply that the substanceP-lir innervation of the dorsalhorn is New York. involved in chemosensorytransmission (Jansco and Kiraly, Kawai, Y., S. Inagaki, S. Shiosaka, E. Senba, K. Takatsuki, M. Sakanaka, 1980).There is evidencethat chemosensoryafferents from mus- K. 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