Physiological Society Symposium Nociceptors as Homeostatic Afferents: Central Processing

Physiological Society Symposium Nociceptors as Homeostatic Afferents: Central Processing Anatomy of primary afferents and projection neurones in the rat spinal dorsal horn with particular emphasis on substance P and the neurokinin 1 receptor A. J. Todd Spinal Cord Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK The dorsal horn of the spinal cord plays an important role in transmitting information from nociceptive primary afferent neurones to the brain; however, our knowledge of its neuronal and synaptic organisation is still limited. Nociceptive afferents terminate mainly in laminae I and II and some of these contain substance P. Many projection neurones are located in lamina I and these send axons to various parts of the brain, including the caudal ventrolateral medulla (CVLM), parabrachial area, periaqueductal grey matter and thalamus. The neurokinin 1 (NK1) receptor on which substance P acts is expressed by certain neurones in the dorsal horn, including approximately 80 % of lamina I projection neurones. There is also a population of large NK1 receptor-immunoreactive neurones with cell bodies in laminae III and IV which project to the CVLM and parabrachial area. It has been shown that the lamina III/IV NK1 receptor-immunoreactive projection neurones are densely and selectively innervated by substance P-containing primary afferent neurones, and there is evidence that these afferents also target lamina I projection neurones with the receptor. Both types of neurone are innervated by descending serotoninergic axons from the medullary raphe nuclei. The lamina III/IV neurones also receive numerous synapses from axons of local inhibitory interneurones which contain GABA and neuropeptide Y, and again this input shows some specificity since post-synaptic dorsal column neurones which also have cell bodies in laminae III and IV receive few contacts from neuropeptide Y- containing axons. These observations indicate that there are specific patterns of synaptic connectivity within the spinal dorsal horn. Experimental Physiology (2002) 87.2, 245 249. Rexed (1952) divided the grey matter of the cat spinal cord into a series of parallel laminae and this scheme which has been extended to other species is now widely used for descriptive purposes. Lamina I corresponds to the marginal layer and lamina II to the substantia gelatinosa, while the remainder of the dorsal horn consists of 4 further laminae (III VI). Despite the obvious importance of the dorsal horn in somatosensory processing, and in particular in pain mechanisms, the complex neuronal and synaptic organisation of the region is still poorly understood. Recent anatomical studies have begun to shed light on the circuitry in laminae I III, and in particular on pathways involving the tachykinin peptide substance P and the neurokinin 1 receptor on which it acts. This review briefly describes the earlier literature concerning primary afferent input to and projections from the dorsal horn, and then summarises recent findings on the expression of NK1 receptors by projection neurones and synaptic inputs to these cells. Primary afferents The dorsal horn is the major site of termination for primary afferent axons. These are distributed in an orderly way based on fibre size and sensory modality. Most small afferents with either fine myelinated (Ad) or unmyelinated (C) axons are nociceptors, and these end predominantly in laminae I and II, although a few reach deeper laminae (Light & Perl, 1979; Sugiura et al. 1987). Some Ad afferents innervate down-hairs and these arborise on either side of the lamina II III border (Light & Perl, 1979). Most large (Ab) cutaneous afferents function as low-threshold mechanoreceptors (but see symposium paper by Lawson in this issue), and each type has a characteristic pattern of Presented at a scientific meeting of the Physiological Society at the University of Bristol in September 2001. Publication of The Physiological Society Email: a.todd@bio.gla.ac.uk 2351

246 A. J. Todd Exp. Physiol. 87.2 arborisation in the deeper laminae (III VI) of the dorsal horn (Brown, 1981). There are several neurochemical markers that can be used to define populations of fine primary afferents, for example many synthesise one or more neuropeptides (Hunt et al. 1992; Lawson, 1992). In the rat it appears that most, if not all, peptidergic afferents contain calcitonin gene-related peptide (CGRP) and since this is restricted to primary afferents in the dorsal horn, antibodies against CGRP can be used to identify these afferents. Many peptidergic somatic afferents contain substance P and physiological studies have suggested that these are all nociceptors (Lawson et al. 1997). Substance P-containing afferents (which include both A and C fibres) end mainly in lamina I and the outer part of lamina II, although some penetrate deeper into the dorsal horn. Some C fibres contain somatostatin and these terminate in the outer part of lamina II (Alvarez & Priestley, 1990; Sakamoto et al. 1999). Approximately half of the C fibres in a somatic nerve do not contain peptides, but most of these can be revealed by their ability to bind the lectin Bandeiraea simplicifolia isolectin B4 (Silverman & Kruger, 1990). Functions of non-peptidergic C fibres are poorly understood; however, it is believed that this population also includes many nociceptors (Guo et al. 1999; Gerke & Plenderleith, 2001). Although there are no intrinsic neurochemical markers which will specifically label myelinated low-threshold mechanoreceptors, these can be identified by transganglionic transport of cholera toxin B subunit (CTb) (Robertson & Grant, 1985). If CTb is injected into a peripheral somatic nerve, it is normally transported only by afferents with myelinated axons and this results in labelling of terminals in lamina I (Ad nociceptors) and in laminae III VI (Ad down-hair afferents and Ab afferents). Projection neurones in the superficial dorsal horn Neurons that project to the brain are most densely packed in lamina I and are also found in the deeper laminae of the dorsal horn (III VI). Cell bodies of spinal projection neurones can be revealed by injecting retrograde tracers into the target nuclei, and this approach was first used by Trevino & Carstens (1975) to reveal spinothalamic tract neurones in cat and monkey. Lamina I neurones have since been shown to project to several sites in the brain including the thalamus, periaqueductal grey matter (PAG), lateral parabrachial area, nucleus of the solitary tract and medullary reticular formation (e.g. Giesler et al. 1979; Menétrey et al. 1982; Cechetto et al. 1985; Menétrey & Basbaum, 1987; Lima & Coimbra, 1988; Burstein et al. 1990; Lima et al. 1991; Todd et al. 2000). These projections are predominantly contralateral. We have estimated the numbers of retrogradely labelled lamina I neurones on the contralateral side of the 3rd lumbar (L3) segment following injection of CTb into various brain sites in the rat (Todd et al. 2000). The largest numbers of labelled cells were seen after injections into the region between the lateral reticular nucleus and the spinal trigeminal nucleus in the caudal ventrolateral medulla Figure 1 Synaptic input from substance P-containing axons to a NK1 receptor-immunoreactive lamina III neurone. a, a confocal image showing a single optical section through part of a dorsal dendrite belonging to a lamina III cell. The section has been reacted with antibodies against NK1 receptor (green), substance P (blue) and CGRP (red). Axons that contain both peptides appear purple. The dendrite (D) receives contacts from several immunostained axons, four of which are marked with numbered arrows. Three of these (1, 3 and 4) contain both peptides and are therefore derived from substance P-containing primary afferents, while the other one (2) contains only substance P. b, the same field after the section was processed to reveal the three primary antibodies with a peroxidase diaminobenzidine technique. c, a low magnification electron micrograph through the same section at a depth approximately corresponding to the confocal image in a. Scale bar, 10 mm. Adapted from Naim et al. (1997).

Exp. Physiol. 87.2 Spinal projection neurones 247 (CVLM) (7.8 11.0 cells per 70 mm transverse section) and the lateral parabrachial area (8.4 9.8 per section). Since the L3 segment is approximately 2.5 mm long, this equates to between 280 and 390 contralateral lamina I neurones labelled from CVLM and between 300 and 350 from the parabrachial area in this segment. More recently, we have injected different tracers into these two regions and found that most retrogradely labelled neurones contained both tracers (A. J. Todd, Z. Puskár & R. C. Spike, unpublished observations). Injections into the PAG resulted in 1.6 3.8 cells per 70 mm section (equivalent to 60 140 cells in the L3 segment) (Todd et al. 2000). In a previous study we had obtained an estimate of 0.3 lamina I neurones per 70 mm section in the lumbar enlargement after injection of CTb into the thalamus, which corresponds to approximately 12 contralateral spinothalamic lamina I neurones per segment (Marshall et al. 1996). There have been two other quantitative studies of the spinothalamic tract in the rat: Lima & Coimbra (1988) obtained a value of 37.5 labelled lamina I neurones on the contralateral side in the combined L3 and L4 segments, while Burstein et al. (1990) found 22 retrogradely labelled lamina I neurones in segments L1, L2 and L3 and 19 such cells in segments L4 and L5. Although there is some variation between these estimates, it is clear that in the rat spinothalamic neurones are greatly outnumbered by those that project to the parabrachial area and CVLM. Substance P acts on the neurokinin 1 (NK1) receptor, and this is present on a restricted population of dorsal horn neurones (Bleazard et al. 1994; Vigna et al. 1994; Brown et al. 1995; Littlewood et al. 1995; Todd et al. 1998). By combining retrograde tracing with immunocytochemistry, we have shown that the NK1 receptor is expressed by approximately 80 % of lamina I neurones that project to the thalamus, periaqueductal grey matter, parabrachial area, or CVLM (Marshall et al. 1996; Todd et al. 2000). Several lines of evidence suggest that neurones with the NK1 receptor respond to noxious stimuli. Henry (1976) found that all dorsal horn neurones in cat spinal cord which were activated by substance P were excited by noxious stimulation, while in the rat it has been shown that most NK1 receptor-immunoreactive neurones in lamina I show internalisation of the receptor (Mantyh et al. 1995) or c-fos expression (Doyle & Hunt, 1999) following acute noxious stimuli. Mantyh et al. (1997) have provided evidence that neurones in the superficial dorsal horn with the NK1 receptor play a critical role in the development of hyperalgesia, since this was dramatically reduced in rats following intrathecal administration of substance P conjugated to the cytotoxin saporin, which selectively destroyed most NK1 receptor-immunoreactive neurones in this region. Less is known about lamina I projection neurones that lack the NK1 receptor; however, within this group we have identified a population of giant cells characterised by the high density of punctate staining with antibody against the glycine receptor-associated protein gephyrin, which outlines their cell bodies and proximal dendrites (Puskár et al. 2001). These cells project to the parabrachial area and most expressed c-fos following intradermal injection of formalin into the ipsilateral hindpaw, which indicates that they respond to acute noxious stimuli. The gephyrin-rich cells are very infrequent; since we found between 5 and 7 per side in each midlumbar segment, they probably make up around 2 % of spinoparabrachial lamina I neurones. Laminae III and IV contain scattered large neurones which are NK1 receptor-immunoreactive and have prominent Figure 2 High magnification electron micrographs of contacts formed by the boutons identified in Fig. 1 and the dendrite which belongs to a lamina III NK1 receptor-immunoreactive neurone. In each case the bouton (numbered as in Fig. 1) is presynaptic to the dendrite (D) at an asymmetrical synapse (between arrows). Scale bar, 0.5 mm. Adapted from Naim et al. (1997).

248 A. J. Todd Exp. Physiol. 87.2 dorsally directed dendrites that arborise within the superficial laminae (Bleazard et al. 1994; Brown et al. 1995; Littlewood et al. 1995). We found that there are approximately 20 of these cells on each side of the spinal cord in the L4 segment, and that they are projection neurones, since virtually all could be retrogradely labelled from the CVLM (Todd et al. 2000). In addition, at least two-thirds of these cells project to the parabrachial area and some to the PAG or thalamus (Marshall et al. 1996; Todd et al. 2000). Inputs to projection neurones We have shown that the NK1 receptor-immunoreactive neurones in laminae III and IV receive numerous synaptic contacts from substance P-containing primary afferents both on their distal dendrites in laminae I II and also in deeper laminae (Naim et al. 1997) (Figs 1 and 2). Since substance P is likely to act on the NK1 receptor through volume transmission (Zoli & Agnati, 1996), we suggested that the presence of synapses is probably more important for mediating the effects of glutamate, which will also be released from these afferents (DeBiasi & Rustioni, 1988). Because the dendrites of these cells branch extensively throughout the dorsal horn they would be in a position to receive synaptic input from various types of afferent. However, we found that they had relatively few synapses from myelinated afferents in laminae III IV (Naim et al. 1998), and very few contacts from either somatostatincontaining or non-peptidergic C fibres in lamina II (Sakamoto et al. 1999). These results indicate that there is a powerful, selective monosynaptic innervation of projection neurones in laminae III and IV which express the NK1 receptor by substance P-containing primary afferents. Less is known about the primary afferent input to lamina I neurones; however, McLeod et al. (1998) reported that many NK1 receptor-immunoreactive dendrites in this lamina received synapses from substance P-immunoreactive axons, and concluded that substance P-containing afferents provided significant input to lamina I projection neurones with the receptor. In agreement with this, we have observed that lamina I projection neurones with the NK1 receptor receive many contacts from axons that contain substance P and CGRP (A. J. Todd, Z. Puskár & R. C. Spike, unpublished observations). Serotoninergic axons from the medulla are thought to contribute to both stimulation-produced and opiate analgesia. Stewart & Maxwell (2000) showed that serotoninimmunoreactive axons form numerous contacts on lamina III/IV neurones with the NK1 receptor, and we have recently found that they also selectively target many lamina I projection neurones with the receptor (Polgár et al. 2002). Part of the action of serotonin is therefore likely to be mediated by a direct effect on spinal projection neurones. There is evidence that the inputs to projection neurones from inhibitory interneurones may also be organised in a selective way. NK1 receptor-immunoreactive neurones in laminae III and IV are heavily innervated by axons containing both neuropeptide Y and GABA (Polgár et al. 1999), which are likely to be derived from local interneurones (Todd & Spike, 1993). This input is specific, since NPY-immunoreactive axons do not make contact with neurones belonging to the post-synaptic dorsal column pathway, which also have cell bodies in laminae III and IV. ALVAREZ, F. J. & PRIESTLEY, J. V. (1990). Anatomy of somatostatinimmunoreactive fibres and cell bodies in the rat trigeminal subnucleus caudalis. Neuroscience 38, 343 357. BLEAZARD, L., HILL, R. G. & MORRIS, R. (1994). The correlation between the distribution of NK 1 receptor and the actions of tachykinin agonists in the dorsal horn of the rat indicates that substance P does not have a functional role on substantia gelatinosa (lamina II) neurons. Journal of Neuroscience 14, 7655 7664. BROWN, A. G. (1981). Organization in the Spinal Cord: The Anatomy and Physiology of Identified Neurones. Springer-Verlag, Berlin. BROWN, J. L., LIU, H., MAGGIO, J. E., VIGNA, S. R., MANTYH, P. W. & BASBAUM, A. I. (1995). Morphological characterization of substance P receptor-immunoreactive neurons in rat spinal cord and trigeminal nucleus caudalis. Journal of Comparative Neurology 356, 327 344. BURSTEIN, R., DADO, R. J. & GIESLER, G. J. (1990). The cells of origin of the spinothalamic tract of the rat: a quantitative reexamination. Brain Research 511, 329 337. CECHETTO, D. F., STANDAERT, D. G. & SAPER, C. B. (1985). Spinal and trigeminal dorsal horn neuron projections to the parabrachial nucleus in the rat. Journal of Comparative Neurology 240, 153 160. DE BIASI, S. & RUSTIONI, A. (1988). Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of the spinal cord. Proceeding of the National Academy of Sciences of the USA 85, 7820 7824. DOYLE, C. & HUNT, S. P. (1999). Substance P receptor (neurokinin-1)- expressing neurons in lamina I of the spinal cord encode for the intensity of noxious stimulation: a c-fos study in the rat. Neuroscience 89, 17 28. GERKE, M. B. & PLENDERLEITH, M. B. (2001). Binding sites for the plant lectin Bandeiraea simplicifolia I-isolectin B 4 are expressed by nociceptive primary sensory neurones. Brain Research 911, 101 104. GIESLER, G. J., MENÉTREY, D. & BASBAUM, A. I. (1979). Differential origins of spinothalamic tract projections to medial and lateral thalamus in the rat. Journal of Comparative Neurology 184, 107 126. GUO, A., VULCHANOVA, L., WANG, J., LI, X. & ELDE, R. (1999). Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. European Journal of Neuroscience 11, 946 958. HENRY, J. L. (1976). Effects of substance P on functionally identified units in cat spinal cord. Brain Research 114, 439 451. HUNT, S. P., MANTYH, P. W. & PRIESTLEY, J. V. (1992). The organization of biochemically characterized sensory neurons. In Sensory Neurones: Diversity, Development and Plasticity, ed. SCOTT, S. A., pp. 60 76. Oxford University Press, New York.

Exp. Physiol. 87.2 Spinal projection neurones 249 LAWSON, S. N. (1992). Morphological and biochemical cell types of sensory neurons. In Sensory Neurones: Diversity, Development and Plasticity, ed. SCOTT, S. A., pp. 27 59. Oxford University Press, New York. LAWSON, S. N., CREPPS, B. A. & PERL, E. R. (1997). Relationship of substance P to afferent characteristics of dorsal root ganglion neurones in the guinea-pig. Journal of Physiology 505, 177 191. LIGHT, A. R. & PERL, E. R. (1979). Spinal termination of functionally identified primary afferent neurons with slowly conducting myelinated fibers. Journal of Comparative Neurology 186, 133 150. LIMA, D. & COIMBRA, A. (1988). The spinothalamic system of the rat: structural types of retrogradely labelled neurons in the marginal zone (lamina I). Neuroscience 27, 215 230. LIMA, D., MENDES-RIBEIRO, J. A. & COIMBRA, A. (1991). The spinolatero-reticular system of the rat: projections from the superficial dorsal horn and structural characterization of marginal neurons involved. Neuroscience 45, 137 152. LITTLEWOOD, N. K., TODD, A. J., SPIKE, R. C., WATT, C. & SHEHAB, S. A. S. (1995). The types of neuron in spinal dorsal horn which possess neurokinin-1 receptors. Neuroscience 66, 597 608. MCLEOD, A. L., KRAUSE, J. E., CUELLO, A. C. & RIBEIRO-DA-SILVA, A. (1998). Preferential synaptic relationships between substance P- immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord. Proceedings of the National Academy of Sciences of the USA 95, 15775 15780. MANTYH, P. W., DEMASTER, E., MALHOTRA, A., GHILARDI, J. R., ROGERS, S. D., MANTYH, C. R., LIU, H., BASBAUM, A. I., VIGNA, S. R., MAGGIO, J. E. & SIMONE, D. A. (1995). Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation. Science 268, 1629 1632. MANTYH, P. W., ROGERS, S. D., HONORE, P., ALLEN, B. J., GHILARDI, J. R., LI, J., DAUGHTERS, R. S., LAPPI, D. A., WILEY, R. G. & SIMONE, D. A. (1997). Ablation of lamina I spinal neurons expressing the substance P receptor profoundly inhibits hyperalgesia. Science 278, 275 279. MARSHALL, G. E., SHEHAB, S. A. S., SPIKE, R. C. & TODD, A. J. (1996). Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat. Neuroscience 72, 255 263. MENÉTREY, D. & BASBAUM, A. I. (1987). Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viserovisceral reflex activation. Journal of Comparative Neurology 255, 439 450. MENÉTREY, D., CHAOUCH, A. & BESSON, J. M. (1982). The origins of the spinomesencephalic tract in the rat: an anatomical study using the retrograde transport of horseradish peroxidase. Journal of Comparative Neurology 206, 193 207. NAIM, M., SPIKE, R. C., WATT, C., SHEHAB, S. A. S. & TODD, A. J. (1997). Cells in laminae III and IV of the rat spinal cord which possess the neurokinin-1 receptor and have dorsally-directed dendrites receive a major synaptic input from tachykinincontaining primary afferents. Journal of Neuroscience 17, 5536 5548. NAIM, M. M., SHEHAB, S. A. S. & TODD, A. J. (1998). Cells in laminae III and IV of the rat spinal cord which possess the neurokinin-1 receptor receive monosynaptic input from myelinated primary afferents. European Journal of Neuroscience 10, 3012 3019. POLGÁR, E., PUSKÁR, Z. & TODD, A. J. (2002). Selective innervation of lamina I projection neurons that possess the neurokinin 1 receptor by serotonin-containing axons in the rat spinal cord. Neuroscience (in the Press). POLGÁR, E., SHEHAB, S. A. S., WATT, C. & TODD, A. J. (1999). GABAergic neurons that contain neuropeptide Y selectively target cells with the neurokinin 1 receptor in laminae III and IV of the rat spinal cord. Journal of Neuroscience 19, 2637 2646. PUSKÁR, Z., POLGÁR, E. & TODD, A. J. (2001). A population of large lamina I projection neurons with selective inhibitory input in rat spinal cord. Neuroscience 102, 167 176. REXED, B. (1952). The cytoarchitectonic organization of the spinal cord in the cat. Journal of Comparative Neurology 96, 415 466. ROBERTSON, B. & GRANT, G. (1985). A comparison between wheatgerm agglutinin and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat. Neuroscience 14, 895 905. SAKAMOTO, H., SPIKE, R. C. & TODD, A. J. (1999). Neurons in laminae III and IV of the rat spinal cord with the neurokinin-1 receptor receive few contacts from unmyelinated primary afferents which do not contain substance P. Neuroscience 94, 903 908. SILVERMAN, J. D. & KRUGER, L. (1990). Selective neuronal glycoconjugate expression in sensory and autonomic ganglia: relation of lectin reactivity to peptide and enzyme markers. Journal of Neurocytology 19, 789 801. STEWART, W. & MAXWELL, D. J. (2000). Morphological evidence for selective modulation by serotonin of a subpopulation of dorsal horn cells which possess the neurokinin-1 receptor. European Journal of Neuroscience 12, 4583 4588. SUGIURA, Y., LEE, C. L. & PERL, E. R. (1987). Central projections of identified, unmyelinated (C) afferent fibres innervating mammalian skin. Science 234, 358 361. TODD, A. J., MCGILL, M. M. & SHEHAB, S. A. S. (2000). Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem. European Journal of Neuroscience 12, 689 700. TODD, A. J. & SPIKE, R. C. (1993). The localization of classical transmitters and neuropeptides within neurons in laminae I-III of the mammalian spinal dorsal horn. Progress in Neurobiology 41, 609 646. TODD, A. J., SPIKE, R. C. & POLGÁR, E. (1998). A quantitative study of neurons which express neurokinin 1 or somatostatin sst 2a receptor in rat spinal dorsal horn. Neuroscience 85, 459 473. TREVINO, D. L. & CARSTENS, E. (1975). Confirmation of the location of spinothalamic neurons in the cat and monkey by the retrograde transport of horseradish peroxidase. Brain Research 98, 177 182. VIGNA, S. R., BOWDEN, J. J., MCDONALD, D. M., FISHER, J., OKAMOTO, A., MCVEY, D. C., PAYAN, D. G. & BUNNETT, N. W. (1994). Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells. Journal of Neuroscience 14, 834 845. ZOLI, M. & AGNATI, L. F. (1996). Wiring and volume transmission in the central nervous system: the concept of open and closed synapses. Progress in Neurobiology 49, 363 380. Acknowledgements Financial support from the Wellcome Trust is gratefully acknowledged.