SYNAPSE 6:344-350 (1990)

Peptide Coexistence in Axon Terminals Within the Smerficial Dorsal Horn of the Eiat Spinal Cord M.B. PLENDERLEITH, C.J. HALLER, AND P.J. SNOW Mammalian Neurobiology Laboratories, Department of Anatomy, University of Queensland, St. Lucia, Queensland, Australia 4072

KEY WORDS

Substance P, Calcitonin gene-related peptide, Protein A-gold,Nociception

The somata of primary sensory neurons have been shown to contain up to four (and possibly more) neuroactive peptides. Although each of these peptides has been separately located in axon terminals within the superficial dorsal horn of the spinal cord, it is not clear whether multiple peptide coexistence is also a feature of terminal varicosities. The aim of this study was to determine whether the peptides substance P (SP) and calcitonin gene-related peptide (CGRP), which are colocalized in the somata of a large number of primary sensory neurons, coexist in the central terminals of these neurons in the spinal cord. The protein A-gold technique of antigen localization was used to screen single boutons in laminae I and I1 of the rats spinal cord for SP- and CGRP-like immunoreactivity a t the ultrastructural level. The results show that SP and CGRP are colocalized within a large number of synaptic boutons in the superficial dorsal horn. Furthermore, evidence was obtained to suggest that both SP and CGRP may be found in the same synaptic vesicle within these boutons. These findings indicate that both SP and CGRP may be coreleased from single terminals in the superficial dorsal horn. This is of considerable interest in view of the reported interaction between SP and CGRP in nociceptive behavioral responses in the rat.

ABSTRACT

INTRODUCTION is effectively expressed (and released) from the neurons A number of neuroactive peptides have been found terminals within the superficial dorsal horn. Alterna-

within the somata of rimary sensory neurons (Cameron et al. 1988; HOl$ elt et al., 1975, 1976; J u et al., 1987, Leah et al., 1985a). For the most part, these eptide-containing neurons are of small diameter and gave unmpelinated axons, which terminate within the superficia laminae of the dorsal horn of the spinal cord (Cameron et al., 1986; Har er and Lawson, 1985; McCarthy and Lawson, 1989; rice, 1985; Sugiura et al., 1986). Consequently it has been suggested that the neuropeptides are involved in synaptic transmission between small-diameter rimary sensory neurons and second-order neurons in t e dorsal horn (Henry, 1982). Most peptide-containing neurons appear to contain more than one peptide; that is, they exhibit peptide coexistence (see Lundberg and Hokfelt, 1986). For example, in recent reports from our laboratory, up to four different peptides have been located within a single feline dorsal root ganglion cell (Cameron et al., 1988; Leah et al., 1985a). The functional significance of peptide coexistence in sensory neurons is far from clear. It has been proposed that the peptide content of a particular neuron may be correlated with its receptor type (Kuraishi et al., 1985).However, no consistent relationship between the modality of a neuron and its peptide content has been demonstrated (Leah et al., 1985b,c). Another possibility is that peptide coexistence is a feature of the neurons somata and that only one peptide

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tively, different synaptic boutons of a single neuron may express different peptides. To investigate these possibilities, we have used a combination of electron microscopy and postembedding immunohistochemistry to screen single synaptic boutons within the rat dorsal horn for the peptides substance P (SP) and calcitonin generelated peptide (CGRP). This enabled us to determine whether the high incidence of coexistencethat these two peptides exhibit within the somata of primary sensory neurons (Gibson et al., 1984; J u et al., 1987; Lee et al., 1985; Matsuyama et al., 1986; Wiesenfeld-Hallin et al., 1984) is expressed in their central terminals.

MATERIALS AND METHODS Three adult male rats (Wistar strain) were anesthetized by an overdose of Nembutal (i.p.1 and perfused through the heart with 0.9% saline containing 0.1% NaN02 followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Lower, lumbar segments of spinal cord were then removed, postfixed for 4 hr, and then sectioned (80 pm, transverse sections) on an Oxford vibratome. These sections were then dehydrated, embedded in L.R. white resin and polymerized at 50°C. Serial ultrathin sections (silver interference Received January 30,1990; accepted in revised form May 21,1990

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color) were then cut with a diamond knife and collected either on gold (200 mesh) grids or on formvar-coated slot grids. For immunohistochemical detection of SP and CGRP we used a modification of the protein A-gold technique of antigen localization (Roth et al., 1978). To begin with, sections were floated on drops of 10% sodium periodate (in H,O) for 5 min in order to etch the embedding medium and thus expose antigenic sites. This was followed by three 5 min washes with antibody dilutant (0.1 M phos hate buffer containing 1%bovine serum albumin). T e sections were then incubated on drops of a commercial1 available olyclonal antisera raised against either P (dilution of1:lOO)or CGRP (dilution of 1:500)in antibody dilutant for 24 hr. Both antisera were raised in rabbits and were purchased from Amersham, (Cambridge, U.K.). Sections were then washed and floated on drops of a 1:lO dilution of a rotein A-gold com lex for 30 min. The protein A-go1c f complex was fres 1 prepared using the tannic acid procedure (Slot and euze, 1985) to yield gold complexes of uniform diameter (6 or 12 nm). In some cases, single sections were double labelled. Following the sequence described above for single labelling the section was incubated in an excess (100 Fg/ml) of free protein A to mask any residual immunoglobulin. The antibody incubation sequence was then repeated with a second antibody and another protein A-gold complex. The different antibody binding sites were delineated by using different sized protein A-gold complexes. Immunohistochemical controls included 1)omission of the first or second antisera from the sequence, 2) incubation of sections with antisera that had been preabsorbed with synthetic SP or CGRP, and 3) replacement of the protein A-gold complex incubation with colloidal gold alone. Following immunohistochemical procedures, sections were stained with methanolic uranyl acetate and lead citrate (Stempak and Ward, 1964) and examined on a Zeiss EM 10 transmission electron microscope.

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RESULTS Electron microscopic examination of sections prior to immunostaining revealed poor ultrastructural preservation of the tissue. This was presumably because we did not postfix the tissue in osmium tetroxide prior to embeddin . Postfixation was omitted because we had consistent y failed to observe SP- or CGRP-like immunoreactivity (LI) following treatment of tissue with osmium. In control sections where the antisera incubation was omitted, where the antisera had been preabsorbed with its native antigen, or where the protein A-gold complex was replaced by colloidal gold alone, no specific immunoreactivity was found. Otherwise we observed a very strong immunoreactivity characterized by a high concentration of gold particles over particular profiles, whereas other profiles showed none. In preliminary single labelling experiments, we found a very similar laminar distribution of SP- and CGRP-LI. Immunoreactivity to both peptide antisera was found concentrated in lamina I and the outer portion of lamina I1 (lamina 11,). Immunoreactivity within laminae I and 11, was found associated with clusters of transversely sectioned unmyelinated axons, with portions of longitudinally

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sectioned unmyelinated axons (Fig. 21, and with terminal profiles forming axodendritic synapses with small and large dendrites (Figs. 2,3). These axodendritic synapses were characteristically asymmetrical, with a prominent postsynaptic density associated with the dendritic membrane. Myelinated axons showing SPor CGRP-LI were only occasionally seen, and no definitive immunoreactive axosomatic contacts were found. Within lamina 11, peptide-containing terminals were found in gomerular-like arrangements where the immunoreactive rofile was surrounded by three or more dendrites. In t e course of this study, however, immunoreactive terminals were only occasionally seen to form synaptic contacts with more than two adjacent dendrites. Within axon profiles and synaptic boutons both SP- and CGRP-LI were found to be associated with large granular vesicles. Clusters of gold particles were seen to overlie large granular vesicles with regions devoid of large granular vesicles exhibiting little immunoreactivity. By incubating single sections in antisera raised against SP and then CGRP and using different-sized colloidal gold-protein A complexes, we were able to distinguish between SP-and CGRP-LI. In these doublelabelled sections we did find axons and synaptic terminals that showed only CGRP- or SP-LI. However, the majority of immunoreactive profiles showed both SPand CGRP-LI (Fig. 1). This pattern of coexistence prevailed irrespective of which size protein A-gold complex was used to label which antibody o r the order in which the antibodies were applied to the section. These results suggest that many synaptic boutons within the superficial laminae exhibit SP and CGRP coexistence. However, examination of control sections on which the secondary antibody had been replaced by antibody dilutant did, on occasion, reveal a very small level of contamination. This was reflected by an occasional gold article of the protein A-gold complex used to visualize t e second antibody in a profile exhibiting heavy immunoreactivity to the first antibody. This type of false-positive result may be due to displacement of a preexisting protein A-gold complex (associated with the first antibody) by the second protein A-gold complex. It is unlikely that the high level of coexistence that we found within single synaptic profiles is due to this very low level of contamination. However, we did attempt to overcome this problem in a second series of experiments in which two adjacent serial sections were incubated in different antisera. After visualization of the antibody binding sites with protein A-gold we were then able to examine single, serially sectioned terminals for SP- and/or CGRP-LI without the problem of protein A-gold displacement. A total of 18 serially sectioned immunoreactive rofiles were located in adjacent sections. Of these, 17 s owed CGRP- and SP-LI (Figs. 2, 31, whereas the other showed only CGRP-LI. These results confirm the above findings showing that many synaptic boutons within laminae I and 11, exhibit SP- and CGRP-LI. A more detailed analysis of the double-labelled single sections revealed an interesting feature. In many instances both SP- and CGRP-LI appeared t o be associated with a single large granular vesicles (Fig. 1).This was supported by evidence from our serial section study. In a number of cases it was possible to locate the same large granular vesicles in two adjacent sections (Fig. 3),

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Fig. 1. A: Electron micrograph of an axon terminal in the outer portion of lamina I1 (lamina IIJ. In this section, a 6 nm-diameter gold probe was used to visualize substance P antibody binding and a 12 nm probe was used to visualize CGRP antibody binding. This terminal contains both SP- and CGRP-like immunoreactivity and is presynaptic to two dendrites (D) as indicated by the prominent postsynaptic densities (long open arrows). Note that peptide immunoreactivity

appears to associated with large granular vesicles. Some large granular vesicles appear to exhibit both SP- and CGRP-like immunoreactivity (short solid arrows) whereas others exhibit neither (short open arrows). Bar = 0.2 pm. B: Higher magnification electron micrograph of a portion of the terminal shown in A to show postsynaptic density and immunoreactive large granular vesicles in more detail. Bar = 0.2 km.

and on occasion it was found that these showed both SPand CGRP-LI (Fig. 3). Finally, it was clear that, whereas many large granular vesicles did exhibit SP- and/or CGRP-LI, other large granular vesicles (within the same terminals) exhibit neither (Fig. 1). DISCUSSION The results of this study show a high level of colocalization of SP- and CGRP-LI within axon terminals in the superficial dorsal horn of the rat spinal cord. That these terminals are in fact the central terminals of primary sensory neurones is suggested by the reported loss of CGRP-LI from the dorsal horn of the rat following dorsal rhizotomy (Gibson et al., 1984). Furthermore, no neurons intrinsic to the dorsal horn have been shown to

exhibit CGRP-LI, even after colchicine treatment (Gibson et al., 1984). We cannot exclude the possibility that some of the CGRP-LI profiles we have observed are the terminals of neurons projecting from supraspinal nuclei. However, as yet there is no evidence for such a projection. A number of studies have examined SP and CGRP coexistence in cells of both the DRG and trigeminal ganglia of the rat (Gibsonet al., 1984;J u et al., 1987;Lee et al., 1985; Matsuyama et al., 1986; Wiesenfeld-Hallin et al., 1984). These have shown that virtually all SP-LI cells also exhibit CGRP-LI. This result is completely consistent with our finding that many synaptic terminals in the superficial dorsal horn showed both SP- and CGRP-LI. However, in the rat DRG, many cells contain-

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Fig. 2. A Electron micrograph of an unmyelinated axon in lamina I. This section was incubated in an antibody raised against CGRP and a 12-nm-diameter gold probe was used to visualize antibody binding. The axon can be seen to contain a number of large granular vesicles that exhibit CGRP-like immunoreactivity (arrows). Bar = 0.5 pm. Inset: Higher magnification micrograph of same axon showing gold particles more clearly. B: The same profile as shown in A in an adjacent section, which was incubated in an antibody raised against SP. Antibody binding was visualized with a 6-nm-diameter gold probe. The terminal exhibits SP-like immunoreactivity also associated with the large gran-

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ular vesicle population (arrows). Bar = 0.5 pm. Inset: Higher magnification micrograph of same axon showing gold particles more clearly. C: Terminal bouton in lamina 11, adjacent to two dendrites (d) one of which exhibits a prominent post-synaptic specialization (open arrow). CGRP-like immunoreactivity is indicated by 12-nm-diameter gold, which appears to be associated only with regions of the terminal containing large anular vesicles. Bar = 0.5 pm. D: The same terminal as shown in Cyut in an adjacent section, which was incubated in an antibody against SP. The 6-nm-diameter gold indicates that this terminal also exhibits SP-LI. Bar = 0.5 pm.

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Fig. 3. A Axon terminal in lamina I showing CGRP-like immunoreactivity as indicated by the 12-nm-diameter gold probes used to visualize antibody binding. Note that immunoreactivity appears to be restricted to a number of large granular vesicles (arrows) in both the . The axon (Ax) and terminal (Ter) region. d, Dendrite. Bar = 0 . 5 ~ mB: same terminal shown in A but in a n adjacent section. This section was inverted prior to immunostaining so that the face exposed to the antibody was that in direct opposition to that stained in the section

shown in A. Note that a number of large granular vesicles that exhibit CGRP-LI in A a p ear also to exhibit SP-LI as indicated by the 6-nm-diameter golfprobe used to visualize antibody binding (arrows). Bar = 0.5 pm. C,D: Higher magnification micrograph of a portion of the terminal shown in A and B, respectively. In at least three cases (arrows) the same large granular vesicle can be identified in both sections and appear to exhibit SP- and CGRP-like immunoreactivity. Bar = 0.2 pm.

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ing CGRP-LI were found not to contain SP-LI (Gibson et al., 1984;J u et al., 1987; Lee et al., 1985; Matsuyama et al., 1986; Wiesenfeld-Hallin et al., 1984). This result is more difficult to reconcile with our analysis of the su erficial dorsal horn, because terminals containin C8RP-LI but not SP-LI were in the minority. Althou this may simply be due to the ualitative nature o f t e present study, another possib e expIanation for this difference is that neurons that contain only CGRP terminate in deeper laminae of the dorsal horn. This suggestion is sup orted by the observation that, whereas CGRP- anfSP-containing DRG cells appear to be of small diameter, those containin CGRP but not SP are usually of large diameter. It is we 1established that the majority of large diameter DRG cells support large diameter myelinated axons (Harper and Lawson, 19851, which terminate in deeper laminae of the dorsal horn (Brown, 1981).On the other hand, small diameter DRG cells appear to be associated with unmyelinated axons (Harper and Lawson, 19851,which terminate in lamina I and I1 (Sugiura et al., 1986). This being the case we might expect to find the majority of primary sensory terminals within the superficial dorsal horn to exhibit both SP- and CGRP-LI. This supposition is further supported by the observation that the population of DRG cells that contain both CGRP and SP appear to be sensitive to the unmyelinated fiber neurotoxin, capsaicin, whereas those containing only CGRP are not (Matsu ama et al., 1986). {he relatively poor ultrastructural reservation we observed in this study did not permit a etailed analysis of the synaptic contacts made by immunoreactive profiles. One surprising finding, however, was the presence of peptide immunoreactivity in glomerular-like profiles; this has not been a common finding in the superficial dorsal horn of the rat (Barber et al., 1979; Breshanan et al., 1984). However, this result is consistent with a recent report by Ribeiro-da-Silva et al. (19891, who demonstrated a significant number of synaptic glomeruli in the rat substantia gelatinosa with SP-LI. Another surprising result of this study was the apparent colocalization of both SP- and CGRP-LI within a single large granular vesicle. Some care should be exercised in this type of interpretation when using the protein A-gold technique of antigen localization because labelling is limited to the surface of the section and, therefore, it is assumed only that immunoreactivity is associated with underlying organelles. However, in certain cases where we could locate the same large granular vesicles in two adjacent sections and erformed antigen localization on opposing faces, we sti 1 found both SP- and CGRP-LI in the same large granular vesicles (Fig. 3). In a recent report, De Biasi and Rustioni (1988)have used a double etching procedure to identify succesfully both SP- and glutamate-like immunoreactivity in single synaptic terminals of the rat spinal cord. This double etching procedure restores immunoreactivity in tissue that has been osmicated and thus exhibits much better ultrastructural detail. In recent preliminary experiments in our laboratory, following the protocol of De Biasi and Rustioni (1988), we successfully localized both SP- and CGRP-LI in single synaptic boutons with much improved ultrastructural resolution. Since the completion of this study, Tuchscherer and Seybold (1989) have published a light microscopic anal-

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ysis of pe tide coexistence within varicosities in the spinal cor of the rat. The results of our electron microscopic study are in a eement with the findin s of Tuchscherer and SeyboE that SP and CGRP are cokcalized in terminal varicosities in the dorsal horn of the rat. Interestingly, these workers provide compelling evidence that varicosities that contain both SP and CGRP are almost entirely of primary afferent origin. The functional significance of the coexistence of SP and CGRP in single terminals within the superficial dorsal horn is still far from clear. Both SP and CGRP have been shown t o be released in the dorsal horn by noxious stimuli applied to the skin (Duggan et al., 1987; Hutchison et al., 1989)and are thought to be involved in synaptic transmission between primary sensory neurons and neurons in the dorsal horn (Henry, 1982). Interestingly, in a behavioral study, Wiesenfeld-Hallin et al. (1984) have shown that the caudally directed biting and scratching response elicited by intrathecal injection of SP was potentiated by CGRP. This enhancement of the effects of SP by exogenously applied CGRP ma be due to CGRP inhibiting breakdown of SP by en opeptidases (Le Greves et al., 19851, CGRP potentiation of SP release (Oku et al., 1987) or both. Whatever the means, it is clear from our finding of SP and CGRP coexistence within single synaptic boutons in the superficial dorsal horn that the potential exists for a similar mechanism to operate when these peptides are released endogenously.

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ACKNOWLEDGMENTS This study was sup orted by the N.H.8zM.R.C. We wish to thank Dr. P. k l s o n for critically reading the manuscript and Ms. D.K. Crook for technical assistance.

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Peptide coexistence in axon terminals within the superficial dorsal horn of the rat spinal cord.

The somata of primary sensory neurons have been shown to contain up to four (and possibly more) neuroactive peptides. Although each of these peptides ...
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