Neuroanatomical localisation of Substance P in the CNS and sensory neurons

Neuropeptides (2000) 34 (5), 256–271 © 2000 Harcourt Publishers Ltd doi: 10.1054/npep.2000.0834, available online at http://www.idealibrary.com on

Neuroanatomical localisation of Substance P in the CNS and sensory neurons A. Ribeiro-da-Silva1,2, T. Hökfelt3 Departments of 1Pharmacology & Therapeutics and 2Anatomy & Cell Biology, McGill University, Montreal, Quebec, Canada 3Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

Summary The anatomical distribution of Substance P (SP) has been investigated since the development of antibodies against it in the 1970s. Although initial studies were performed with antibodies that also recognised the other endogenous neurokinins, most of the initial descriptions are surprisingly still valid today. In this review, we provide an integrated overview of the pathways containing SP in the central and peripheral nervous systems. The highest densities of SP immunoreactivity occur in the superficial dorsal horn of the spinal cord, in the substantia nigra and in the medial amygdaloid nucleus. In the peripheral nervous system, SP occurs in high concentrations in small diameter primary sensory fibres and in the enteric nervous system. SP is extensively co-localised with classical transmitters and other neuropeptides. In the spinal cord, SP immunoreactive axonal boutons are preferentially presynaptic to neurons expressing the SP receptor, suggesting that the neurokinin acts at a short distance from the release site. In contrast, in the periphery, the situation probably differs in the autonomic ganglia, where the targets are directly innervated by SP, and in other peripheral territories, where SP has to diffuse through the connective tissue to reach the structures expressing the receptor. © 2000 Harcourt Publishers Ltd

INTRODUCTION The first description of Substance (SP) immunoreactivity in the central and peripheral nervous systems of rat and cat was published in 1975 (Hökfelt et al., 1975). Subsequently, two reports provided a comprehensive description of the localization of SP-immunoreactive (IR) cell bodies and fibre systems in the central nervous system (CNS) (Ljungdahl et al., 1978; Cuello and Kanazawa, 1978), and several studies described SP immunoreactivity in the peripheral nervous system (PNS) (see e.g. Dalsgaarg et al., 1982; Cuello et al., 1978; Costa et al., 1980). These initial reports utilised antibodies raised against the carboxy-terminal sequence of the peptide, shared by all neurokinins, and thus would be expected to cross-react with the other tachykinis. However, subse-

Received 11 July 2000 Accepted 15 July 2000 Correspondence to: Alfredo Ribeiro-da-Silva, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, room 1325, Montreal, Quebec, Canada H3G 1Y6 Tel. +514 398; 3619; Fax +514 398; 6690; E-mail [email protected]

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quent studies using antibodies that recognized only SP showed that most of the localisations originally described contained bona fide SP immunoreactivity. One of the main features of SP immunoreactivity is its co-localisation with other transmitter/modulator substances. In particular, the co-localisation of SP with classical transmitters is so widespread that the question was posed whether a neuropeptide like SP can occur in the absence of a classical transmitter (Hökfelt et al., 2000). This article will present an overview of the distribution of the SP in the central and peripheral nervous systems and will outline the major pathways in which this neurokinin is involved. We will also discuss the colocalisation of SP with classical transmitters such as 5hydroxytryptamine (5-HT) and with other neuropeptides such as calcitonin gene-related peptide (CGRP), as well as ultrastructural features of SP-IR boutons and their morphological relationship with post-synaptic targets expressing the substance P receptor. Because of limited space, our reference list is restricted, and readers are often referred to other publications for a more comprehensive review.

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B 15.20 mm

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Fig. 1 Diagrammatic representations of coronal plane sections of the central nervous system of the rat illustrating the distribution of SP immunoreactivity, as assessed in a recent study (Ribeiro-da-Silva et al., 2000) using a bi-specific anti-SP/anti-HRP monoclonal antibody. The scale of the terminal immunostaining was based on a visual assessment of the density, and not intensity, of the immunostaining. Values represent distance of diagrams from the interaural line. Diagrams were modified from Ribeiro-da-Silva et al., (2000), and from Paxinos’ atlas (Paxinos and Watson, 1986). 3V, third ventricle; ac, anterior commissure; Acb, Nucleus accumbens; AcbC, core of the nucleus accumbens; AcbSh, shell of the nucleus accumbens; Amb, nucleus ambiguus; AOB, Accessory olfactory bulb; AOn, anterior olfactory nucleus; cb9, cerebellar lobule 9; cb 10, cerebellar lobule 10; DC, dorsal column; DG, dentate gyrus; DR, dorsal raphe nucleus; DTg, dorsal tegmental nucleus; EPl, external plexiform layer of olfactory bulb; f, fornix; Cg, cingulate cortex; CPu, caudate-putamen; dmn10, dorsal motor nucleus, © 2000 Harcourt Publishers Ltd

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Fig. 1 continued vagus nerve; Gl, glomerular layer, olfactory bulb; GP, globus pallidus; GrA, granular cell layer of accessory olfactory bulb; H, hilus, hippocampal formation; Hb, habenulla; HDB, horizontal limb of the diagonal band of Broca; Hypoth., hypothalamus; I-IIA, laminae I and outer lamina II, spinal cord or trigeminal subnucleus caudalis; ICj, islands of Calleja; IGr, inner granular cell layer, olfactory bulb; IP, interpeduncular nucleus; IPl, internal plexiform layer, olfactory bulb; LHb, lateral habenular nucleus; LC, locus coeruleus; LPB, lateral parabrachial nucleus; LPO, lateral preoptic area; LSN, lateral spinal nucleus; MG, medial geniculate nucleus; MHb, medial habenular nucleus; Mi, mitral cell layer, olfactory bulb; MnMPO, medial preoptic area; MnR, median raphe nucleus; MPB, medial parabrachial nucleus; MS, medial septal nucleus; nbm, nucleus basalis magnocellularis; OT, olfactory tubercle; ox, optic chiasma; PAG, periaqueductal gray; Par, parietal cortex; PP, peripeduncular nucleus; Pr5, principal sensory trigeminal nucleus; PV, paraventricular thalamic nucleus; RMg, raphe magnus nucleus; Rbd, rhabdoid nucleus; SC, superior colliculus; SI, substantia innominata; SN, substantia nigra; Sol, nucleus of the solitary tract; Sp5C, spinal trigeminal nucleus, pars caudalis; Sp5I, spinal trigeminal nucleus, pars interpolaris; Sp5O, spinal trigeminal nucleus, pars oralis; VDB, ventral limb of diagonal band of Broca; VP, ventral pallidum; VTA, ventral tegmental area. Neuropeptides (2000) 34(5), 256–271

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Neuroanatomical localisation of Substance P

METHODS OF STUDY Although SP immunoreactivity is easily detected in nerve fibres and terminals, its detection in cell bodies is considerably more difficult to achieve, as the peptide generally does not occur in high concentrations in nerve cell bodies. The first study detected SP perikaryal immunoreactivity in one CNS region only, the medial habenula (Hökfelt et al., 1975). A subsequent study detected it in very few CNS locations (Cuello and Kanazawa, 1978). However, the use of colchicine allowed the demonstration of SP immunoreactivity in over 30 CNS regions, including the spinal cord and several locations in the brain stem (Ljungdahl et al., 1978). The use of colchicine can be criticised, as there is evidence that it can turn on genes and therefore might lead to false positive results. Nevertheless, these neurons have the capacity to synthesize SP under these conditions. Furthermore, in our experience, with today’s antibodies and immunocytochemical approaches, SP immunoreactivity is easily detected in cells bodies of all the regions in which it was described following colchicine treatment, although sometimes masked by the heavy terminal and fibre staining. In situ hybridization has allowed the validation of data obtained with immunocytochemistry on the localisation of SP in nerve cell bodies. However, this approach detects a message that is common to SP and neurokinin A (NKA). In the rat, this is not a real limitation as almost all neurons with the message produce precursors that generate both SP and NKA (Carter and Krause, 1990), leading to a virtually complete co-localisation of SP and NKA. SP immunoreactivity occurs in both local circuit neurons and in long pathways. The identification of SP immunoreactivity in specific neuronal pathways has been possible mainly by a combination of either nerve lesions or tract-tracing methods with SP immunostaining. An interesting example of the application of this approach was the experimental demonstration that SP immunoreactive (IR) fibres in the skin and most of the SP-IR fibres in the dorsal horn are of primary sensory origin, following electrolytical destruction of the trigeminal ganglia and surgical interruption of the peripheral nerves (Cuello et al., 1978). DISTRIBUTION OF SP IMMUNOREACTIVITY IN THE CNS The present review focuses on the CNS of the rat. However, species differences are indicated when they are of particular significance. Figure 1 provides a diagrammatic representation of SP-IR fibre and cell body distribution in the CNS of the male Wistar rat, based on a study (Ribeiro-da-Silva et al., 2000) applying a bi-specific

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monoclonal antibody recognizing both SP and the marker (horseradish peroxidase – HRP) (Suresh et al., 1986). As each of the antibody molecules has only one binding site for SP and one for HRP, the detected signal is proportional to the peptide levels in the tissue. Furthermore, although it is directed against the C terminal of SP, it does not recognize neurokinin B in the concentrations used in immunocytochemistry; however, it does recognise equally well SP and NKA (McLeod et al., 2000). This is not a real limitation as in the rat virtually all neurons that express SP co-express NKA (Carter and Krause, 1990).

Olfactory regions In the main olfactory bulb, SP immunoreactivity occurs as scattered fibres and varicosities, except in the internal plexiform and granular cell layers, where they are more abundant. In the accessory olfactory bulb, there is a dense network of SP-IR fibres (Fig. 1A). Most likely, these systems are intrinsic to the olfactory bulbs. SP immunoreactivity occurs with a rather low density in most of the olfactory tubercule, but is more intense in the innermost part. In the area of the medial forebrain bundle and islands of Caleja (Fig. 1D), SP immunoreactivity is intense.

Cerebral Cortex In rat, the innervation of the neocortex by SP-IR fibres is not abundant. The only region in which a well-defined SP-IR fibre network is detected is the cingulate cortex at rostral levels (Fig. 1B–D). However, the fibre density is low, and only isolated fibres and varicosities are seen in other neocortical regions. If conditions are optimised and the detection level increased to the maximum, some non-pyramidal neurons in layers II and IV–VI appear immunostained, and the fibre network becomes more prominent, even in parietal regions (Fig. 2A). These results were obtained independently in both of our laboratories, applying different protocols, and would indicate that SP-IR cortical systems in rat are more prominent than originally thought. Further evidence that our results are genuine originates from in situ hybridization studies that have revealed signals for the mRNA of the precursor of SP/NKA in layers II and IV–VI of the cortex (Warden and Young, 1988). Neocortical fibres and cell bodies immunoreactive for SP seem to be more prominent in other species such as cat (Conti et al., 1992) and human (Mai et al., 1986; Pioro et al., 1990). Therefore, it is possible that there is not much SP in the rat cortex under basal conditions but that it may be upregulated under special circumstances.

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Temporal Cortex (Layer V)

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Hippocampal Formation (CA1)

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Fig. 2 Examples of SP immunoreactivity in cortical areas in the rat CNS. A – Parietal cortex, layer V; note the sparse fibre network and the small non-pyramidal perikarya. B – CA1 of hippocampal formation; note that immunostaining is limited to an occasional non-pyramidal cell body and very sparse fibres. Scale bars = 50 µm.

Hippocampal formation (Fig. 1G)

Basal ganglia (Figs. 1C–G)

The hippocampal formation is usually cited as an example of a CNS area where there is a mismatch between the SP innervation and its receptor. This is due to the fact that, although immunoreactivity for the substance P receptor (NK-1r) occurs throughout the hippocampal formation and is strong in the hilus of the dentate gyrus, there is scarce SP immunoreactivity in this region, as only scattered fibres are detected when conventional approaches are applied. However, we were able to detect weakly immunoreactive cell bodies in the pyramidal cell layer and stratum oriens (Fig. 2B) and a sparse network of SP-IR fibres. As the cell body distribution exactly matches that of neurons expressing the mRNA for the SP/neurokinin A precursor (Warden and Young, 1988), our immunocytochemical data are likely to be genuine. In cat (Vincent et al., 1981) and in human (Mai et al., 1986; Pioro et al., 1990), hippocampal SP systems seem to be more developed than in rodents.

SP immunoreactivity in the caudate-putamen corresponds to numerous very fine fibres and varicosities. SP-IR cell bodies have been detected with the use of colchicine (Ljungdahl et al., 1978), but can also be detected without it. These perikarya are known to be the origin of a pathway that terminates in the substantia nigra and gives off collaterals to the striatum itself. In the nucleus accumbens, the immunostaining pattern for SP is similar to that encountered in the caudate-putamen; however, SP immunoreactivity is very strong in some areas. In the globus pallidus (Fig. 1F), SP immunoreactivity consists of a dense network of small fibres and varicosities that seems to outline dendritic processes of unlabelled neurons, as originally described (Ljungdahl et al., 1978).

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Basal forebrain cholinergic nuclei In the basal forebrain cholinergic nuclei (Figs. 1E–F), intense SP immunoreactivity is seen. However, in some © 2000 Harcourt Publishers Ltd

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regions, such as the medial septum, SP-IR fibres and varicosities are weakly labelled. In contrast, in the nucleus basalis magnocellularis (Fig. 3A) and in the substantia innominata, the plexus of SP-IR fibres is rather dense and seems to directly innervate the cholinergic neurons.

In the periaqueductal gray (Figs. 1H–I), SP immunoreactivity is rather strong and consists of a high density of small varicosities and a few cell bodies. In the raphe nuclei (Fig. 1I), SP–IR fibres are detected at a rather low density in most nuclei, and are particularly noticeable in the median raphe nucleus.

Ventral pallidum

Pons

In most of the ventral pallidum (Figs. 1D–E), SP immunoreactivity is particularly intense and dense.

In the parabrachial nuclei (Fig. 1J), SP immunoreactivity is distributed across the entire extent of the nuclei and is particularly strong in some areas. SP immunoreactivity is intense in the locus coeruleus (Fig. 1J). In the reticular formation of the pons, there are sparse to moderate levels of SP immunoreactivity. An important species difference has recently been observed using double in situ hybridisation. In fact, a co-localisation of the message for SP and the 5-HT transporter has been detected in the human dorsal raphe nucleus (Sergeyev et al., 1999), confirming a previous immunocytochemical study that had suggested the occurrence of such colocalisation based on indirect evidence (Baker et al., 1991). Such co-localisation has never been detected in the rat and may help explain the role of SP in depression and the therapeutic action of NK-1 receptor antagonists in this condition (Hökfelt et al., 2000; Kramer et al., 1998). In the trigeminal principal sensory nucleus (Fig. 1J) there is little SP immunoreactivity; in the trigeminal motor nucleus (Fig. 1J) there are just a few SP-IR fibres.

Amygdala In the amygdaloid complex (Fig. 1G), SP immunoreactivity is particularly intense in the caudal and dorsal part of the medial amygdaloid nucleus, with lower immunoreactivity levels in other regions. Diencephalon In the preoptic area (Fig. 1F), SP-IR fibres and terminals occur with medium to high densities. In the hypothalamus (Fig. 1G), SP immunoreactivity occurs with moderate abundance in several nuclei, and is scarce in others. In the thalamus (Fig. 1G), most nuclei contain low levels of SP immunoreactivity. The ventrobasal complex displays almost no SP-IR structures. A few SP-IR fibres are detected in some nuclei, particularly at caudal levels in the parafascicular nucleus. More abundant SP immunoreactivity is present in midline regions at more rostral levels. In the habenula (Fig. 3B), the medial habenular nucleus displays a high density of small cell bodies with strong SP immunoreactivity, but only in its dorsal part. In the lateral habenular nucleus there is a rather dense network of SP-IR fibres and terminals. Mesencephalon The most striking observation in the mesencephalon is the very high density of SP innervation of the substantia nigra (Figs. 1H and 3C), particularly in the pars reticulata. In the ventral tegmental area, low levels of SP immunoreactivity are detected (Fig. 1H). In the interpeduncular nucleus (Figs. 1H and 3C), SP immunoreactivity is abundant in the lateral part. In the superior colliculus (Figs. 1H–I), there are several small neurons with SP immunoreactivity, and fibres and terminals, which occur mainly in two bands (see Figs. 1H–I). Only isolated SP–IR fibres are detected in most of the inferior colliculus (Fig. 1I), although the most peripheral parts had a moderately dense network of SPIR fibres. © 2000 Harcourt Publishers Ltd

Medulla oblongata The spinal trigeminal nucleus displays only sparse SP immunoreactivity in the subnuclei oralis and interpolaris (Figs. 1K–L). In the trigeminal subnucleus caudalis (Fig. 1M), SP immunoreactivity is particularly concentrated in lamina I and outer lamina II, as in the dorsal horn of the spinal cord (see below); like in the spinal cord, a few immunoreactive cell bodies were detected. In the solitary tract nucleus (Figs. 1K–M), SP immunoreactivity is observed in a dense network of fibres and varicosities throughout the nucleus. In the rostral part of the nucleus ambiguus (Fig. 1L), SP innervation is rather intense around the nucleus, but just a few fibres penetrate its core. In the dorsal motor nucleus of the vagus, there is rather abundant SP innervation. In the hypoglossal nucleus, SP-IR varicosities can be seen around the motoneurons. Of the medullary raphe nuclei, most display SP immunoreactivity. The raphe nuclei are particularly important, as they possess SP-IR cell bodies that project to the caudal medulla and to the spinal cord (see below). Neuropeptides (2000) 34(5), 256–271

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MHb

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Fig. 3 Examples of SP immunoreactivity in several CNS regions. A – In the nucleus basalis magnocellularis (NBM), note the dense innervation by SP-IR fibres and boutons, which are clustered on unstained neuronal cell bodies and dendrites. B – In the habenula, SP immunoreactivity was mostly absent from the ventral part of the medial habenular nucleus (MHb), but occurred as a cluster of densely packed cell bodies (arrowhead) in the dorsal part of the nucleus. SP immunoreactivity was intense in the lateral habenular nucleus (LHb) and of moderate intensity in the paraventricular thalamic nucleus (PV). C – In the ventral part of the mesencephalon, observe the extremely dense and intense immunostaining in the substantia nigra (SN). Note also that in the interpeduncular nucleus (IP) SP immunoreactivity is intense in the lateral part, whereas in the medial part numerous small cell bodies can be observed (arrowheads). Scale bars = 100 µm.

Cerebellum (Figs. 1J–L)

Spinal cord (Fig. 1M)

SP immunoreactivity in the cerebellum is limited to isolated fibres.

SP immunoreactivity is particularly intense in lamina I and outer lamina II, but decreases substantially in inner

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Fig. 4 Diagrammatic representation of SP-containing neuronal pathways in the CNS and PNS of the rat. The brain is shown in horizontal view. Neurons from the caudate-putamen (Cpu) send a high number of axons through the internal capsule (ic) to end in the substantia nigra (SN), particularly to the pars reticulata but also to the pars compacta. These fibres send collaterals that end in the neostriatum, in the entopeduncular nucleus (EP) and, most likely, around the cholinergic neurons of the nucleus basalis magnocellularis (NBM). In the SN, the SP-IR fibres establish repetitive synapses on dopaminergic neurons (represented on right side of diagram). SP-IR cell bodies, located in the medial habenula (MHb), project to the lateral habenula (LHb), to the interpeduncular nucleus (IP) and to the dorsal raphe (DR). SP-IR neurons from the raphe magnus nucleus (RMg), which co-localize 5-HT and other peptides, project to the spinal trigeminal nucleus (SpV), the ventral horn (VH) and, to a minor extent, the dorsal horn (DH) of the spinal cord. SP-IR neurons in the PAG have a similar projection to the SpV and VH of spinal cord. Neurons in the medial amygdala (Amg) project to the ventral amygdala and, through the stria terminalis (st), to other levels (see below). SP-IR neurons in the bed nucleus of the stria terminalis (BST) branch locally and also project to the medial preoptic area of the hypothalamus (Hy). Some of the fibres that terminate in the cingulate cortex (Cx) and in the lateral septum (Lat. Spt) originate from the laterodorsal tegmental nucleus (LDTg). Some fibres in the raphe magnus (RMg) originate from the cuneiform nucleus (Cnf). The solitary nucleus (Sol) receives fibres from SP-IR perikarya in the sensory ganglia of the VII, IX and X cranial nerves. The peripheral branches of these nerves terminate around blood vessels, in viscera and glands. The central branches of the SP-IR neurons of the trigeminal and sensory ganglia terminate in the superficial layers of the trigeminal subnucleus caudalis (SpV) and dorsal horn of spinal cord, respectively; the peripheral branches of these neurons terminate around blood vessels, glands and epidermis of the skin. Some peripheral branches of SP-containing sensory neurons in spinal ganglia terminate in prevertebral sympathetic ganglia, where they are presynaptic to noradrenergic neurons; other branches terminate as free ending in the wall of the gastro-intestinal tract. Diagram modified from Cuello et al. (1985)

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C Fig. 5 Ultrastructural features of SP immunoreactivity in axonal boutons in the dorsal horn of the rat spinal cord, using a bi-specific antiSP/anti-HRP monoclonal antibody. A – SP immunoreactivity in the central bouton of a synaptic glomerulus (a structure known to be of sensory origin) in lamina II. In an attempt to restrict the subcellular localisation of the reaction product, a rather high glutaraldehyde concentration was used and the sections were not counterstained. The reaction product can be observed on the large granular vesicles (LGV), but some light deposits can also be seen in between the agranular vesicles and outlining the plasma membrane and the mitochondria. Small punctual deposits (arrows in inset) of reaction product are in the size range of the small agranular vesicles. The inset represents an enlargement of the outlined area. Modified from Ribeiro-da-Silva et al. (1989). Scale bar = 0.2 µm. B – Example of the colocalization of SP (dense precipitate) and enkephalin (silver grains of photographic emulsion) in an axon terminal that establishes an asymmetric synapse (arrows) with a dendritic spine in lamina I; the bi-specific anti-SP/anti-HRP antibody was combined with an antienkephalin internally radiolabelled monoclonal antibody (modified from Ribeiro-da-Silva et al. (1991). Scale bar = 0.5 µm. C – The association between an NK-1 receptor immunoreactive dendrite (NK–1r+) and a SP-IR bouton (SP+). Note that the SP-IR bouton establishes an asymmetric synapse (arrow) on the NK-1 receptor-IR dendrite. Asterisks represent non-immunoreactive boutons apposed and/or presynaptic to the same dendrite. Note the absence of NK-1 receptor immunoreactivity (represented by silver-gold particles – arrowheads) from the synaptic sites in this preparation from lamina III. Modified from McLeod et al. (1998). Scale bar = 1 µm. Neuropeptides (2000) 34(5), 256–271

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lamina II (Ribeiro-da-silva et al., 1989). In lamina III, SP immunoreactivity is further reduced, and is represented mostly by fibres crossing towards deeper laminae. In laminae IV–V, there are clusters of SP-IR fibres and boutons separated by areas with sparse immunoreactivity (Ruda et al., 1986). There is also considerable SP immunoreactivity in the lateral spinal nucleus and around the central canal, extending dorsally along the medial edge of the dorsal horn. In the ventral horn, there is a loose network of SP-IR fibres and boutons. Contrary to common belief, it should be stated that not all SP immunoreactivity in the superficial laminae of the spinal dorsal horn is of sensory origin. Multiple dorsal rhizotomies and capsaicin treatments have never been able to deplete all SP immunoreactivity. In agreement with this, SP-IR cell bodies have been identified in laminae I and II, as well as V of the spinal cord, both with immucytochemistry (Ljungdahl et al., 1978; Ribeiro-daSilva et al., 1991) and in situ hybridisation (Warden and Young, 1988). Furthermore, although most descending SP-containing systems from the brain stem terminate in the ventral horn (Gilbert et al., 1982; Hökfelt et al., 1978), some may terminate in the superficial laminae of the dorsal horn. An interesting feature of SP-IR cell bodies in the spinal dorsal horn is that most, if not all, co-localise enkephalin immunoreactivity (Ribeiro-da-Silva et al., 1991). An example of SP/enkephalin localisation in axon terminals in the rat superficial dorsal horn is shown in Figure 5B. In cat, but not rat, some of the SP-IR cell bodies and axon terminals in lamina I co-localise GABA immunoreactivity (Ma and Ribeiro-da-Silva, 1995). SUBSTANCE P-CONTAINING PATHWAYS IN THE CNS Curiously, most studies that sought to identify SP pathways in the CNS were performed in the 1970’s and 80’s. This is unfortunate, as the use of the novel techniques available to us now would certainly fill gaps in our knowledge that still persist. A symptom of this situation is the fact that Figure 4, which provides a diagrammatic representation of SP pathways, was modified from diagrams by Cuello and collaborators dating from the 1980s (Cuello et al., 1982; Cuello et al., 1985). In the cerebral cortex, most SP-containing systems are probably intrinsic, although the evidence for this is indirect. The number of SP-IR cell bodies that can be detected with both immunocytochemistry and in situ hybridisation seems to be sufficient to justify the sparse fibre network of most cortical regions. However, in the cingulate cortex, a region where SP-IR fibres are more abundant, some of the SP innervation is of extrinsic origin (Fig. 4). This was demonstrated by Paxinos et al. (1978), using microknife lesions, and was originally © 2000 Harcourt Publishers Ltd

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thought to originate from a level caudal to the mesencephalon. Subsequent studies have shown that the brain stem laterodorsal tegmental nucleus (LDTg; Fig. 4) projects to the cingulate cortex (Sakanaka et al., 1983), making it the likely source of the extrinsic neocortical SP-IR fibres. Lesions of the septal area produced a reduction of what was defined as SP immunoreactivity in the hippocampal formation (Vincent and McGeer, 1981); however, as SP-IR fibres in the hippocampal formation are sparse in the rat and the neurokinin B (NKB) system is considerably more prominent (Lucas et al., 1992), this system may correspond in the rat to a NKB rather than to a SP system. The SP-containing neurons of the caudate-putamen have been shown to project massively to the substantia nigra (Fig. 4) (Hong et al., 1977; Kanazawa et al., 1977; Brownstein et al., 1977; Jessell et al., 1978), where the axon terminals have been shown to be presynaptic to the dopaminergic neurons and at least in part to co-localise GABA (Bolam and Smith, 1990). Tracing methods have provided evidence that striato-nigral neurons send axon collaterals to the striatum itself (Fig. 4). Because of the morphological properties of the cell bodies (Lee et al., 1997), it is likely that these represent the SP-containing population. Striato-nigral neurons would also send collaterals to the entopenduncular nucleus (Paxinos et al., 1978; Kanazawa et al., 1980), to the cholinergic neurons of the basal forebrain, especially those in the nucleus basalis magnocellularis (NBM) (Henderson, 1997), and to the globus pallidus as suggested in the review by Reiner and Anderson (1990). Neurons expressing the message for the SP/NKA precursor have been shown to project to the ventral pallidum and nucleus accumbens (Napier et al., 1995). Although the medial amygdaloid nucleus displays one of the heaviest concentrations of SP-IR terminals in the CNS, most of this immunoreactivity is of local origin (Fig. 4), as demonstrated following multiple knife cuts (Emson et al., 1978). However, the amygdala appears to send fibres into the stria terminalis (Sakanaka et al., 1981). Some of the SP-IR fibres in the medial preoptic area seem to originate from the bed nucleus of the stria terminalis (Paxinos et al., 1978). The SP-IR fibres in the interpreduncular nucleus clearly originate from the medial habenula (Artymyshyn and Murray, 1985; Emson et al., 1977; Groenewegen et al., 1986), which also projects to the dorsal raphe nucleus (Fig. 4) (Neckers et al., 1979). The SP-IR spinal cord projections were identified in the late 1970’s by a combination of immunocytochemistry and lesions (Hökfelt et al., 1978), and were later confirmed in the early 1980’s by a combination of tracttracing methods and immunocytochemistry. SP-IR projections (Fig. 4) have been described from the Neuropeptides (2000) 34(5), 256–271

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periaqueductal grey, in neurons that also co-localise cholecystokinin (Skirboll et al., 1983), and from the raphe magnus (Bowker et al., 1981), in neurons that also co-localise 5-HT (Hökfelt et al., 1978, Chan-Palay et al., 1978) and thyrotropin-releasing hormone (Johansson et al., 1981). The medullo-spinal neurons projecting to the spinal cord, which co-localise SP and 5-HT, occur in several nuclei besides the raphe magnus, namely the raphe pallidus, raphe obscurus, pars alpha of the nucleus reticularis gigantocellularis and nucleus intrafascicularis hypoglossi (Hökfelt et al., 1978). Descending pathways co-localizing SP with 5-HT and other peptides have been confirmed in several species, such as cat and primates (for recent review see Hökfelt et al. (2000). An interesting recent development was the identification in both rat and primate that some of the neurons co-localising SP and 5-HT also co-localise glutamate/aspartate immunoreactivity (Nicholas et al., 1992). Substance P in the peripheral nervous system It is now well established that SP occurs in a subpopulation of primary sensory neurons, which release the neurokinin in both central and peripheral endings. If the role of SP released in the spinal cord and other sensory areas is to participate in the transmission/modulation of pain-related information, that released in peripheral ends plays a role in cutaneous antidromic phenomena, such as antidromic plasma extravasation and vasodilation, or the regulation of sympathetic ganglia activity (for review see Cuello [1987] and Elfvin et al. [1993]). SP immunoreactivity occurs in a subpopulation of primary sensory neurons that have their cell bodies located in spinal (Hökfelt et al., 1975) and trigeminal (Cuello et al., 1978) ganglia. The SP-IR neurons in spinal ganglia send their central processes to the dorsal horn of the spinal cord (Fig. 4), particularly to lamina I and outer lamina (Ljungdahl et al., 1978; Cuello and Kanazawa, 1978; Ribeiro-da-Silva et al., 1989) but also to lamina V (Ruda et al., 1986) and, in small quantities, through the dorsal columns to terminate in the dorsal column nuclei (Cuello et al., 1978; Costa et al., 2000). SP-IR perikarya in the trigeminal ganglion send their central processes to the caudal spinal trigeminal nucleus (Fig. 4) (Cuello et al., 1978; Del Fiacco and Cuello, 1980). The peripheral branches (Fig. 4) terminate in the skin and in deeper structures such joints and viscera. Of the fibres that terminate in the skin, some penetrate the epidermis and others end around blood vessels, hair follicles or glands in the dermis (Hökfelt et al., 1975; Dalsgaard et al., 1983). A triadic arrangement of a blood vessel, a terminal of a SP-IR fibre and a mast cell is frequently encountered (see Fig. 6) (Ribeiro-da-Silva et al., 1988). There is also evidence that some SP-IR visceral afferents have their Neuropeptides (2000) 34(5), 256–271

cell bodies located in the sensory ganglia of the VII, IX and X cranial nerves and send their central branches to the solitary tract nucleus (Cuello et al., 1985). An interesting arrangement has been described for SP-IR sensory fibres that innervate the gastrointestinal tract, as they have collaterals that establish synapses on post-ganglionic autonomic neurons in prevertebral sympathetic ganglia (Fig. 4) (Matthews et al., 1987) (for reviews see Cuello (1987) and Elfvin et al. (Elfvin et al., 1993)). This type of arrangement allows sensory information originating from the gastro-intestinal tract to directly influence the sympathetic system, suggesting the presence of an extra level of sensory-autonomic integration which has the unique feature of bypassing the CNS (Cuello, 1987). On primary sensory neurons, SP was found to be co-localised with the classical transmitter glutamate (Battaglia and Rustioni, 1988; De Biasi and Rustioni, 1988) and with neuropeptides such as calcitonin generelated peptide (CGRP) (Wiesenfeld-Hallin et al., 1984) and galanin (Ju et al., 1987). In the gastro-intestinal tract, SP-IR sensory fibres are abundant in the submucous and myenteric plexuses (Costa et al., 1980; Matthews and Cuello, 1982; Matthews and Cuello, 1984). Curiously, SP immunoreactivity has also been detected in some nerve cell bodies in enteric ganglia of the submucous and myenteric plexuses (Costa et al., 1980; Costa et al., 1996). These perikarya receive close appositions from SP-IR terminals, although classical synapses are seldom observed (Llewellyn-Smith et al., 1989). ULTRASTRUCTURAL STUDIES AND FUNCTIONAL CONSIDERATIONS Since the initial ultrastructural studies of SP immunoreactivity in the CNS (Chan-Palay and Palay, 1977; Pickel et al., 1977), most studies using an immunoperoxidase approach have characterized staining in axonal terminals as being located on large granular vesicles (LGV) and in between the agranular vesicles. In contrast, studies using post-embedding immunogold protocols have restricted the signal to the LGV (De Biasi and Rustioni, 1988; Merighi et al., 1989). The exclusive storage and nonsynaptic release of neuropeptides from LGV would support a radically different mode of neuron to neuron communication when compared to classical transmitters, as release of the peptide-containing LGV would occur outside synaptic sites and would require a different firing pattern for the neuron (for recent review see (Hökfelt et al., 2000; Bean et al., 1994). When it comes to SP, however, there are several arguments that challenge this hypothesis, lending support to a study from the late 1970s that showed that SP immunoreactivity was highest in a cell fraction rich in agranular synaptic vesicles © 2000 Harcourt Publishers Ltd

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A1

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A2

M

M V

V

B

M

Fig. 6 SP immunoreactivity in the skin of the rat lower lip. An example of a triadic arrangement composed of SP-IR varicosities (arrows), a mast cell (M) and a blood vessel (V). A – SP-IR varicose fibres surround a mast cell (two focal planes at the light microscopy level, scale bar = 20 µm). B – Note the intense immunostaining of two varicose fibres. Agranular synaptic vesicles can clearly be observed in the upper terminal, which displays relatively less immunostaining. The lower axon displays several extensive continuous varicosities (scale bar = 0.5 µm).

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(Cuello et al., 1977). Firstly, the amount of reaction product that occurs in terminals when using a direct immunocytochemical approach with bi-specific monoclonal antibodies is so high that it cannot be explained by precipitate diffusion from LGV, as there is no signal amplification (i.e. there is one SP molecule per HRP molecule bound to antibody). Secondly, using the above antibody and optimising the immunostaining conditions, images suggesting the occurrence of SP immunoreactivity in agranular vesicles were observed (Fig. 5A) (Ribeiroda-Silva et al., 1989). Thirdly, after optimising the conditions for post-embedding immunogold staining, the gold particles are not restricted to the LGV (Ribeiroda-Silva, unpublished observations). Studies using post-embedding immunogold staining on ultrathin cryosections are required to clarify this issue. An important issue is whether, once released, SP acts at a distance from the site of release, or only in the cells apposed to the terminals or at a very short distance from them. This point has been address in studies involving the simultaneous detection of SP and NK-r immunoreactivities at the light and ultrastructural levels. Initial observations detected mismatches between the distribution of the SP innervation and the cells expressing the NK-1r (Liu et al., 1994), however, a recent quantitative study using a new anti-NK-1r antibody revealed that in laminae I-III of the rat spinal cord SP-IR boutons preferentially innervate dendrites immunoreactive for the NK-r (Fig. 5C) (McLeod et al., 1998). These results have been confirmed by another group, who have shown using confocal microscopy that NK-1r-IR neurons in layers III-IV of the dorsal horn receive considerably more appositions from SP-IR boutons than neurons that expressed choline acetyltransferase immunoreactivity (Naim et al., 1997). Curiously, such preferential innervation of neurons expressing the NK-1r was still present when the SP-IR fibres occured in an ectopic location through genetic manipulation in a transgenic mouse model (McLeod et al., 1999). Further evidence in support of the concept that SP-containing fibres preferentially innervate neurons that are SP targets was obtained by combining intracellular physiological characterisation and labelling of cat dorsal horn neurons with SP immunocytochemistry at the EM level. This demonstrated that SP-IR boutons preferentially innervate neurons that possess a specific type of nociceptive response that has been interpreted as being mediated by SP (De Koninck et al., 1992; Ma et al., 1996). The above data allows us to conclude that, in the spinal cord at least, the SP innervation of other neurons does not occur at random. In fact, SP-containing boutons seem to preferentially innervate neurons upon which the peptide acts, indicating the occurrence of a specific targeting mechanism. Our data also suggests that SP would act normally at Neuropeptides (2000) 34(5), 256–271

a short distance from the release site. The CNS is full of examples of SP/NK-1 receptor mismatches which indicate that the signalling of the neuron is complex, particularly for pathways such as striato-nigral, which sends collaterals to areas rich in NK-1 receptors such as the striatum itself. In the striatum, SP and the main transmitter would act on the areas containing SP receptors, but only the main transmitter (e.g. GABA) would act in areas and neurons devoid of SP receptors. The above issue is discussed at length in a recent review (Ribeiro-da-Silva et al., 2000). In the skin, SP-IR boutons in the epidermis and terminating around vessels, glands or hair follicles display, like their CNS counterparts, a few LGV and many agranular synaptic vesicles (Fig. 6). However, synapses are only observed in autonomic ganglia (Matthews et al., 1987; Llewellyn-Smith et al., 1989). In contrast with the CNS, in which SP probably acts preferentially in a quasi-synaptic manner, in the skin SP has to diffuse in the connective tissue to get to the targets. Final Remarks This review stresses that after an initial period in which the anatomical study of SP pathways received particular attention, interest in the subject has declined, despite the many gaps which exist in our knowledge and the extremely powerful approaches that are now available to address these gaps. The still current view that SP acts in a diffuse manner, like a ‘neurohormone’, has made detailed anatomical studies unfashionable and switched scientific attention to studies of its receptors. However, recent evidence strongly suggests that, for a proper understanding of SP’s function in the nervous system, we need to know both the anatomical details of SP pathways and the precise location and innervation pattern by SP of the neurons expressing the NK-1r. Grant support: Dr. A. Ribeiro-da-Silva acknowledges grant support from the Canadian MRC (grants MT-12170 and MOP-38093). Dr. Hökfelt acknowledges support from the Swedish Medical Research Council (grant 04X-2887).

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