Anat Embryol (1990) 181 : 271-280 Anatomy and Embryology �9 Springer-Verlag 1990

The immunocytochemical distribution of seven peptides in the spinal cord and dorsal root ganglia of horse and pig

Adalberto Merighi 1, 2 Satyabrata Kar 1, Sally J. Gibson 1, Silvana Ghidella 2, Armando Gobetto 2, Saverio M. Peironc 2, and Julia M. Polak 1 1 Department of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, United Kingdom 2 Dipartimento di Morfofisiologia Veterinaria, Universitfi di Torino, Torino, Italy

Accepted October 18, 1989

Summary. The distribution of calcitonin gene-related pep- tide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal poly- peptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig.

In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concen- trated in laminae I III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of immunoreactive nerve fibres was noted in the lumbosacral segments of the spinal cord, which was particu- larly exaggerated in the case of VIP immunoreactivity. In the horse, some CGRP-, somatostatin- or tachykinin-im- munoreactive cell bodies were present in the dorsal horn. In the pig, cells immunoreactive for somatostatin, enkepha- 1in or NPY were noted in a similar location.

In the ventral horn most motoneurones were CGRP- immunoreactive in both species. However, in pig many other cell types were CGRP-immunoreactive not only in the ventral horn, but also in laminae V-VI of the dorsal horn.

With the exception of enkephalin and NPY immunore- activity, which was not seen in pig dorsal root ganglia, all peptides studied were localised to neuronal cell bodies and/or fibres in the dorsal root ganglia. In both species, immunolabelled cell bodies were observed in ganglia from cervical, thoracic, lumbar and sacral levels, with the excep- tion of VIP-immunoreactive cells that were detected only in the lumbosacral ganglia. Numerous CGRP- and tachyk- inin-immunoreactive cell bodies were visualised in both spe- cies, while the cells immunolabelled with other peptide anti- sera were much lower in number.

In both species, immunostaining of serial sections re- vealed that a subset of CGRP-immunoreactive cells co-ex- pressed tachykinin, galanin or somatostatin immunoreac- tivity. In the horse some enkephalin-immunoreactive cells were also CGRP positive and occasionally combinations of three peptides, e.g. CGRP, tachykinin and galanin or CGRP, tachykinin and enkephalin were identified.

The results obtained suggest that the overall pattern of distribution of peptide immunoreactivities is in general

Offprint requests to: Sally J. Gibson

agreement with that so far described in other mammals, although some species variations have been observed, par- ticularly regarding the presence of immunoreactive cell bod- ies in the dorsal horn of the spinal cord.

Key words: Spinal cord - Dorsal root ganglia - Horse - Pig - Neuropeptides

Introduction

The distribution of neuroactive peptides has been widely studied in the nervous system and there now exists extensive literature on the topographical mapping of neuropeptides in the spinal cord and in sensory ganglia, including the dorsal root ganglia (for reviews see Gibson and Polak 1986; Ruda et al. 1986; Dalsgaard 1988).

The dorsal horn of the spinal cord (laminae I VI ac- cording to the classification of Rexed 1952, 1954) is known to be the site of termination of the central projections of primary afferent fibres derived from sensory neurones in the dorsal root ganglia (Brown 1982), but it also contains intrinsic spinal neurones and supraspinal descending fibres (Tracey 1985). The list of peptides known to be present in primary afferent fibres and in dorsal root ganglion cells includes calcitonin gene-related peptide (CGRP) (Gibson et al. 1984a; Wiesenfeld-Hallin et al. 1984; Lundberg et al. 1985), dynorphin (Kawatani et al. 1984; Basbaum 1985), enkephalin (Senba et al. 1982; Kawatani et al. 1984; Rop- pollo et al. 1984), galanin (Ch'ng et al. 1985; Skofitsch and Jacobowitz 1985 a), mammalian bombesin/gastrin-releasing peptide (GRP) (Panula et al. 1983; Fuxe 1983), neurokin- in A (Dalsgaard et al. 1985), somatostatin (H6kfelt et al. 1975a, 1976; Tuchsherer and Seybold 1985), substance P (H6kfelt et al. 1975b, 1976; Cuello et al. 1976, 1978; Ljund- h/il et al. 1978) and vasoactive intestinal polypeptide (VIP) (Lundberg et al. 1978; H6kfelt et al. 1982; Gibson et al. 1984b). The same peptides, with the exception of CGRP, have been identified in neuronal cell bodies in the spinal cord. In addition, other peptides, such as neurotensin (Sey- bold and Elde 1982; DiFiglia et al. 1984), and neuropepti- de Y (Allen et al. 1983; Lundberg et al. 1984), which are not found in primary sensory neurones, have also been de- tected in intrinsic spinal neurones of the dorsal horn.

The ventral horn of the spinal cord (laminae VII-IX)

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is mainly occupied by the different groups of motoneurones, but also by intrinsic spinal neurones and by collaterals of afferent and descending fibres (see Kernell 1986). The inter- mediolateral cell columns of the thoracic and lumbar spinal cord contain the cell bodies of the preganglionic sympathet- ic neurones and fibres of afferent and supraspinal origin (Molander et al. 1984). Peptide innervation of the ventral horn and intermediolateral cell columns is far less abundant than in the dorsal horn. Nevertheless, fibres and/or nerve cell bodies (besides the motoneurones) containing enkepha- lin (H6kfelt et al. 1977), NPY (Allen et al. 1983), somatos- tatin (Johansson et al. 1984; Schroder 1984), substance P (H6kfelt et al. 1975b, 1977; Cuello et al. 1976) and VIP (Gibson et al. 1984b) have been described also in the ventral horn and the intermediolateral cell columns. The most abundant peptides in the motoneurones are CGRP (Gibson et al. 1984a) and galanin (Ch'ng et al. 1985).

Little is known about the function of the spinal grey matter surrounding the central canal (lamina X), but it re- ceives nociceptive information (Light and Perl 1979) and is a peptide rich area in which the majority of peptides detected in the dorsal horn were also demonstrated (Gibson et al. 1981).

Despite the numerous data available on the distribution of peptides in the spinal cord and dorsal root ganglia, their functional role is still far from clear. Evidence suggests that some peptides such as CGRP (Goodman and Iversen 1986), enkephalin (Jessel and Iversen 1977; Fields et al. 1980), so- matostatin (Salt and Hill 1983) and substance P (Lembeck 1953; H6kfelt et al. 1975b, 1977; Salt and Hill 1983) are involved in the processing of primary sensory information. However, the same peptides are also believed to regulate the activity of ventral horn motoneurones (Nicoll et al. 1980; Zieglg/insberger and Tulloch 1979). Likewise, VIP seems to be involved in the transmission of sensory stimuli from pelvic viscera, but also in the regulation of autonomic function (DeGroat 1987; Kawatani et al. 1983; Gibson and Polak 1986). The function of other substances such as ga- lanin, GRP, and NPY is even more uncertain, although it seems that GRP and NPY are involved in the regulation of autonomic and visceral function (Anand and Bloom 1984; Burks et al. 1985), while galanin is probably involved in the regulation of both somatosensory and motor activity (R6kaeus 1987).

The above reported studies have mainly included inves- tigations of laboratory rodents, cats, monkeys and man. However a detailed study on peptide distribution in the spinal cord and dorsal root ganglia of other animal species has not been carried out and this may prove useful in under- standing the evolution of peptide-containing pathways and provide further insight on the functional role of these mole- cules.

The aim of the present investigation, therefore, was to map the distribution of peptides that are associated with sensory processing (CGRP, enkephalin, galanin, somatos- rain, tachykinins and VIP) as well as those associated with autonomic nerves (NPY, VIP) and motoneurones (CGRP, enkephalin, galanin, tachykinins) in the spinal cord of horse and pig. Dorsal root ganglia from both species were also examined for the presence of peptide-containing sensory neurones and colocalisation of immunoreactants. In so do- ing, a comparison between the results obtained in horse and pig tissues could be made with those so far described in other mammals.

Materials and methods

Tissue preparation

Spinal cord and dorsal root ganglia from different segmen- tal levels (Cz-C3, Tz-T3, L2-L3, $2-$3) were studied from animals of both sexes. Tissues from young adult horses (n = 20) and pigs (n = 6) were collected in a public abattoir immediately after death. The cord was cut into slices ap- proximately 0.5-1 cm thick and the larger of the dorsal root ganglia were divided into two or three pieces. The material was then fixed by immersion in Bouin's fluid. Some specimens were collected from young piglets (n = 8, 4-8 kg) that were perfused transcardially with Bouin's fluid after being anaesthetised with an overdose of sodium pentobarbi- tone. Following fixation for 6-24 h depending upon the size of the sample, slices of the spinal cord and dorsal root ganglia were washed in phosphate buffered-saline (PBS). Some samples of spinal cord were then sectioned directly with a Vibratome and 20-30 gm free-floating sections were further processed for immunocytochemistry. The remaining tissues were either transferred into PBS containing 15% (w/v) sucrose and 0.01% sodium azide at + 4 ~ for at least 24 h before processing to cryostat blocks, or dehy- drated, cleared and embedded in paraffin. Serial sections of dorsal root ganglia (6-8 ~tm) and 10-20 ~tm transverse sections of the cord were collected on poly-L-lysine (Sigma Chemicals, USA) coated slides (Huang et al. 1983) and pro- cessed for immunocytochemistry.

Immunocy tochemistry

A. Tissue pretreatment

To unmask antigenicity and reduce non-specific back- ground, paraffin sections were treated, prior to immuno- staining, with 0.06% trypsin (Sigma Chemicals, USA) or with 0.003% subtilisin (Sigma Chemicals, USA; Protease type VII or "Nagarse" protease) dissolved in 0.15 M Tris- buffered saline (TBS) pH 7.4 for 15-60 rain at room tem- perature (Fiocca et al. 1987).

B. Immunostaining procedures

Sections were incubated with primary antisera at optimal titre overnight at + 4 ~ C and then immunostained with the avidin-biotin-peroxidase method (ABC, Vector, UK). The peroxidase reaction was developed using the glucose oxi- dase-nickel-DAB method (Shu et al. 1988). Sections were subsequently rinsed in PBS, dehydrated and mounted in DPX (Raymond A. Lamb, UK).

C. Colocalisation studies

Coexistence of peptides within dorsal root ganglion cells was studied on reverse face (Gibson et al. 1984a), conven- tional serial cryostat or paraffin sections.

Antisera

Well-characterised rabbit polyclonal antisera against CGRP (Gibson et al. 1984a), enkephalin (McGregor et al. 1984), galanin (Ch'ng et al. 1985), neurokinin A (Valentino etal. 1986), NPY (Gibson etal. 1984c), somatostatin (McGregor et al. 1984), substance P (McGregor et al. 1984) and VIP (Gibson et al. 1984b) were used in this study. Sub-

stance P antiserum showed a partial cross-reactivity with neurokinin A as demonstrated by liquid phase absorption. The pattern of immunostaining with substance P and neu- rokinin A antisera was very similar in both species; there- fore, the material detected with these two antibodies is re- ferred to as tachykinin-immunoreactive throughout the text.

The enkephalin antiserum employed in this study mainly recognised methionine-enkephalin, but immunostaining was partially reduced after absorption with high concentra- tions of leucine-enkephalin. It is therefore possible that this antiserum may exhibit some cross-reactivity with dynor- phin, since the latter contains the leucine-enkephalin se- quence at its N terminus.

Controls

Immunocytochemical controls consisted of liquid phase ab- sorption of primary antisera with corresponding and inap- propriate antigens, their substitution with normal serum, omission of goat anti-rabbit biotinylated antibodies or avi- din-biotin-peroxidase complex in the ABC procedure.

Results

Peptide distribution

Spinal cord

Dorsal horn (laminae I-VI). In both species, immunoreac- tivity for each peptide studied was observed in all segmental levels of the spinal cord examined (cervical, thoracic, lum- bar, sacral). Only VIP immunoreactivity appeared to be particularly more concentrated in the lumbosacral segments of the cord than in more rostral segments. Immunostaining for all the peptides studied was observed in varicose fibres that formed a dense plexus in laminae I and II of the dorsal horn. Immunoreactive fibres were found predominantly concentrated in lamina II (the substantia gelatinosa). The intensity of immunostaining varied greatly when different primary antisera were employed: e.g. CGRP-, enkephalin-, tachykinin-, somatostatin- and NPY-immunoreactive fibres were very abundant, while labelling with the galanin and VIP antisera immunostained consistently fewer fibres (Fig. 1 A-G). Some CGRP-, tachykinin-, somatostatin- or galanin-immunoreactive fibres were observed in Lissauer's tract. Scattered CGRP-, tachykinin-, somatostatin-, NPY- and enkephalin-immunoreactive fibres were detected in la- minae I I I ~ I .

In laminae I - I I of the horse dorsal horn, some cell bod- ies were immunoreactive for CGRP (Fig. 1 A) or tachykin- ins (Fig. 1 H), while in laminae IV-V a few somatostatin immunolabelled cells were observed. In laminae I I - I I I of the pig cord some cells were immunoreactive for enkephalin (Fig. 1B), NPY (Fig. I D) or somatostatin. In the pig CGRP-immunoreactive cells were detected in laminae V-VI (Fig. 1 I).

Ventral horn and intermediolateral cell columns (laminae VII-IX). In the ventral horn of both species scattered CGRP-, somatostatin-, tachykinin-, NPY- and enkephalin- immunoreactive fibres were observed in all levels of the spinal cord studied (Fig. 2C, F). Most motoneurones were CGRP-immunoreactive in both species (Fig. 2A). CGRP-

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immunoreactive cells in the ventral horn were particularly evident in the pig where, besides the motoneurones, other positive cells were observed in laminae VII-IX.

Immunoreactive axons were detected in both species as- sociated with the intermediolateral cell columns (Fig. 2C, D, G). Here, the staining was very intense in the pig spinal cord, where enkephalin (Fig. 2 C), NPY- (Fig. 2 D) and ta- chykinin-immunoreactive fibres formed a prominent plexus around the presumptive autonomic preganglionic neurones of thoracic and lumbar segments.

Grey matter surrounding the central canal (lamina 32). Nu- merous CGRP-, somatostatin-, tachykinin- and NPY-im- munoreactive fibres in both species were observed in the spinal grey matter dorsal to the central canal, where they formed a discrete plexus (Fig. 2E). Scattered CGRP- and tachykinin-immunoreactive fibres were observed also ven- tral to the central canal. In the sacral segments of the pig a striking feature was a group of cells in the ventral grey commissure which were immunoreactive for CGRP (Fig. 2B).

Dorsal root ganglia

CGRP- and tachykinin-immunoreactive fibres within the dorsal root ganglia were very abundant in both species. A few fibres were also occasionally stained with galanin and somatostatin antisera. In the horse, scattered NPY- immunoreactive fibres were also detected. These observa- tions were confirmed at all segmental levels.

All peptides under study, except NPY in the horse and NPY and enkephalin in the pig, were localised to cell bod- ies. Immunoreactive cells were generally of small and/or medium size.

A large number of CGRP-immunoreactive cells were present. Immunostaining with tachykinin antisera also re- sulted in visualisation of moderate numbers of positive cells. By contrast only a few somatostatin-, galanin-, enkephalin- (in the horse only) and VIP-immunoreactive cells were ob- served (Fig. 3 A-D).

Segmental differences among ganglia were not ob- viously evident, with the exception of VIP-immunoreactive cell bodies that were observed only in the lumbosacral gan- glia.

Colocalisation of peptides within dorsal root ganglion cells

Examination of pairs of serial sections incubated with dif- ferent primary antisera revealed some cells that contained more than one peptide. This observation was confirmed at each of the four segmental levels studied and there was no evidence of differences in the pattern of colocalisation when ganglia from various levels were examined. Demon- stration of two different antigens within the same neuronal cell body was a common finding; cells containing CGRP + tachykinin, CGRP + galanin, CGRP + somatostatin and ta- chykinin+galanin were observed in both species. In the horse, colocalisation of CGRP and enkephalin was also observed. Examination of series of three serial sections, after immunostaining with different primary antisera, some- times showed coexistence of CGRP + galanin + tachykinin (Fig. 3 E-G) or CGRP + tachykinin + enkephalin (Fig. 3 H - J) immunoreactivities in the same cell.

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Fig. 2A-G. Peptide immunoreactivities in the ventral horn, intermediolateral cell columns and gray matter surrounding the central canal of the horse and pig. A CGRP-immunoreactive motoneurones in the lumbar spinal cord of pig ( x 240). B CGRP-immunoreactive neurones in the region of the central canal (*) in the pig sacral spinal cord ( x 90). C Enkephalin (ENK)-immunoreactive fibres in the intermediotateral cell column (imI) and in lamina VII of the pig thoracic spinal cord. *, Central canal ( x 100). D NPY immunoreactivity in the region of the intermediolateral cell column (iml) of the pig thoracic spinal cord ( x 80). E Somatostatin (SOM)-immunoreactive fibres in the region around the central canal (*) of the horse lumbar spinal cord ( x 80). F Tachykinin (TACHY)-immunoreactive fibres in the cervical ventral horn of the horse ( x 80). G Tachykinin (TACHY) immunoreactivity in the intermediolateral cell column (iml) of the horse thoracic spinal cord ( x 80)

Fig, 1A-I. Peptide distribution in the dorsal horn of the horse and pig spinal cord. A CGRP immunoreactivity in the horse lumbar dorsal horn. A positive celI body in lamina I (rectangle) is shown in the inset ( x 38; inset x 450). B Enkephalin (ENK) immunoreactivity in the thoracic spinal cord of the pig, a positive cell body (rectangle) in lamina III is shown in the inset ( x 140; inset x 450). C Galanin (GAL)-immunoreactive fibres in the pig lumbar spinal cord (x 80). D NPY immunoreactivity in the thoracic spinal cord of the pig. An immunoreactive cell body (rectangle) is shown in the inset (x 140; inset x450). E Horse spinal cord at the cervical level after immunostaining with somatostatin (SOM) antiserum ( x 38). F VIP-immunoreactive fibres in the horse sacral spinal cord. Positive fibres form a bundle at the border between the dorsal horn (dh) and the lateral funiculus (/j). Lt, Lissauer's tract ( x 128). G Tachykinin (TACHY) immunoreactivity in the horse sacral spinal cord (x 51). H Two tachykinin-immunoreactive cell bodies in the lamina II of the horse sacral dorsal horn ( x 450). I Cell bodies immunoreactive for CGRP in the lamina VI of the pig sacral spinal cord ( x 210)

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Fig. 3A-J . Peptides in the horse and pig dorsal root ganglia. A CGRP-immunoreactive cell bodies and fibres in a cervical dorsal root ganglion of pig ( x 210). B Galanin (GAL)-immunoreactive cells in a thoracic dorsal root ganglion of pig ( x 330). C Tachykinin (TA CHY)- immunoreactive cells in a lumbar ganglion of pig ( x 330). D VIP-immunoreactive nerve cell bodies in a sacral dorsal root ganglion of horse (x 330). E -G Serial sections through a horse lumbar dorsal root ganglion. Colocalisation of CGRP (E), galanin (F, GAL) and tachykinin (G, TACHY) in a single cell (arrow), two other cells (asterisks) are immunoreactive for both CGRP and tachykinin (x370). H - J Serial sections through a cervical dorsal root ganglion of horse. Colocalisation of CGRP (H), tachykinin (I, TACHY) and enkephalin (J, ENK) in the same cell is clearly seen ( x 370)

Discussion

Peptide distribution

The present investigation describes the localisation of seven different peptides within the spinal cord and the dorsal root ganglia of the horse and pig in order to compare the results obtained in these two species with the data available in other mammalian species. The results concerning each pep- tide will be therefore discussed separately and compared with previous reports.

Calcitonin gene-related peptide (CGRP)

Previous studies on the distribution of CGRP immunoreac- tivity in the spinal cord and dorsal root ganglia of several mammals, including man, provided evidence that immuno- reactive fibres in the dorsal horn have exclusively an ex- traspinal source from cell bodies of primary sensory neu- rones in the dorsal root ganglia (Gibson et al. 1984a; Har- mann et al. 1988; McNeill et al. 1988). The results of the present investigation confirm the existence of numerous CGRP-immunoreactive cell bodies in the dorsal root gan- glia from both species; moreover, the pattern of distribution of immunoreactive fibres in the spinal cord of horse and pig is in general agreement with the above reported observa- tions. However, a striking feature appears to be the ex- istence of CGRP-immunoreactive cells in the superficial la- minae of the horse dorsal horn, which have not been re- ported previously in the other species so far studied, includ- ing man. Furthermore, CGRP-immunoreactive neurones have been detected in laminae V VIII and X of the pig spinal cord, in addition to motoneurones. Since no CGRP- immunoreactive cell bodies in the dorsal horn have been visualised in the rat, despite colchicine pretreatment (Gib- son et al. 1984a), it seems likely that real interspecies differ- ences exist in the distribution of immunoreactive cell bodies within the spinal cord. The functional significance of these findings is far from clear. However, the morphology of immunoreactive neurones in the horse dorsal horn and in pig laminae V-X suggest that they are spinal interneurones that may be involved in the formation of local circuits.

Enkephalin

The results of the present investigation showed that the general pattern of distribution of enkephalin-immunoreac- tive fibres in the spinal cord of horse and pig is similar to that previously reported in other mammals. Some spinal intrinsic neurones in the pig dorsal horn were also identified as enkephalin-immunoreactive in the present study. This is consistent with the view that most enkephalin immunore- activity in the spinal cord originates from intrinsic spinal neurones, since deafferentation and spinal transection pro- duced no detectable changes in the amount and/or distribu- tion of this peptide (Del Fiacco and Cuello 1980; Jancso et al. 1981; Ruda et al. 1983). On the other hand, in the horse a few cell bodies in the dorsal root ganglia were la- belled with the enkephalin antiserum. The presence of enke- phalin-immunoreactive cell bodies in the dorsal root ganglia has been a matter of contention; however, other authors have been able to detect some positive nerve cells in the dorsal root ganglia of rat (Senba et al. 1982), cat (Kawatani et al. 1984) and monkey (Roppollo et al. 1984). These ob- servations would indicate that in the pig spinal cord enke-

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phalin immunoreactivity has exclusively an intrinsic origin, while in the horse some immunoreactive fibres in the spinal cord may derive from cell bodies in the dorsal root ganglia. In this study colchicine pretreatment was not employed; hence, one can not exclude the existence of neurones in the spinal cord and/or in the dorsal root ganglia that were unstained because of their low antigen content. However, the effect of colchicine administration on enkephalin immu- noreactivity may be unpredictable considering that, in the rat, pretreatment with colchicine was found useful for the demonstration of cell bodies in the spinal cord, while no effect was seen on the number of positive cells in the dorsal root ganglia (Senba et al. 1982).

Galanin

Galanin has previously been demonstrated in the spinal cord and dorsal root ganglia of the rat and pig by radioim- munoassay and immunohistochemistry (R6kaeus et al. 1984; Ch'ng et al. 1985; Skofitsch and Jacobowitz 1985a; Melander et al. 1984). The distribution in the two species was similar, although galanin-containing cell bodies were demonstrated only in the rat spinal cord. The pattern of galanin-immunoreactive fibres in the spinal cord of horse and pig resulting from this investigation is largely in accor- dance with the above studies. Positive cell bodies were not detected in the horse and pig spinal cord, therefore suggest- ing the existence of interspecies differences with the rat.

Neuropeptide Y (NPY)

NPY immunoreactivity has been detected within the spinal cord of both species, appearing to be concentrated in the dorsal horn, where, in the pig, numerous immunoreactive nerve cells were also noticed, In contrast to the other pep- tides, NPY was not seen in the cells of the dorsal root ganglia of either species. These observations are consistent with those indicating that NPY is intrinsic to the spinal cord in other mammals so far examined (Allen et al. 1983; Lundberg etal. 1984; Gibson etal. 1984c; Ruda etal. 1986).

Somatostatin

The pattern of distribution of somatostatin immunoreactiv- ity in the spinal cord and dorsal root ganglia was very similar in the horse and pig. These observations are in agreement with the results in monkeys (Forssmann 1978; Burnweit and Forssmann 1979) and rats (H6kfelt et al. 1975a, 1976; Dalsgaard et al. 1981 ; Hunt et al. 1981 ; Jo- hansson et al. 1984; Schroder 1984), although immunoreac- rive cell bodies in the spinal cord were more frequently observed in these species.

Tachykinins

The pattern of distribution of tachykinin immunoreactivity in the spinal cord and dorsal root ganglia of the horse and pig is very similar to that described in rat (see for example H6kfelt et al. 1975b, 1976; Lundberg et al. 1978; Dalsgaard etal. 1985; Tuchsherer and Seybold 1985), guinea pig (Lundberg et al. 1978; Matthews and Cuello 1982), cat (H6kfelt et al. 1975b; Leah et al. 1985a, b) and man (Charnay et al. 1983). Tachykinin-immunoreactive cell bodies were visualised in the horse spinal cord only; how- ever, colchicine administration was found necessary for the

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demonstration of substance P immunoreactivity within the spinal cord in most of the above reported studies, therefore suggesting that the same experimental treatment might be necessary to confirm the existence of immunoreactive cell bodies in the pig spinal cord.

Vasoactive intestinal polypeptide (VIP)

The pattern of VIP immunoreactivity in the two species studied here is remarkably similar to that described pre- viously in the rat, cat, marmoset monkey and man (Anand etal. 1983, 1984; DeGroa t 1983, 1987; Kawatani etal . 1983; Gibson et al. 1984b). It is interesting to note that VIP immunoreactivity in the spinal cord and dorsal root ganglia of both species is concentrated at the lumbosacral level. In the sacral spinal cord of the cat the majority of VIP is derived from pelvic visceral afferents, as was demon- strated combining immunocytochemistry with horseradish peroxidase retrograde tracing (Kawatani et al. 1983; De- Groat 1987), which accounts for increased levels of immu- noreactivity in lumbosacral segments of the cord (Gibson et al. 1984 b, 1986). In the view of these experimental results it could be speculated that also in the horse and pig the concentration of VIP immunoreactivity at lumbar and sac- ral levels might be related to a VIP-ergic input from pelvic viscera.

Peptide colocalisation in the dorsal root ganglia

Colocalisation of different peptides within cells of the dorsal root ganglia was observed in both species. In most cases two different peptides were detected in the same cell, i.e. C G R P + tachykinin, C G R P + galanin, C G R P + somatosta- tin and tachykinin + galanin. The combinations reported in this study have already been described in other mammals. For example, depending upon the species, a total or subto- tal coexistence between C G R P and substance P has been discovered in guinea pigs and rats (Gibson et al. 1984a; Wiesenfeld-Hallin et al. 1984; Gibbins et al. 1985, 1987; Lundberg et al. 1985; Skofitsch and Jacobowitz 1985b; Wanaka et al. 1986). Coexistence of C G R P and galanin was described in the rat (Ju et al. 1987) and the combination C G R P + somatostatin was described in rats (Ju et al. 1987) and humans (Dalsgaard et al. 1989). Finally, most galanin- immunoreactive primary sensory neurones in the rat also appeared to contain substance P (Ju et al. 1987; Skofitsch and Jacobowitz 1985 a). In the horse it was possible to dem- onstrate the colocalisation of C G R P and enkephalin and also cells containing more than two peptides, i.e. C G R P + tachykinin + galanin and C G R P + tachykinin + enkephalin. The localisation of enkephalin (an opioid peptide) to prima- ry sensory neurones in the horse, even though in a very small population of cells, may be rather surprising; never- theless, it is in accordance with the observation of leucine- enkephalin-immunoreactive neurones in the rat dorsal root ganglia (Senba et al. 1982) and of dynorphin (another opioid peptide) immunolabelled cells in the guinea pig (Gib- bins et al. 1987). The combination C G R P + tachykinin + enkephalin observed in horse might therefore correspond to the combination C G R P + substance P + dynorphin de- scribed in afferents projecting to the pelvic viscera and air- ways in the guinea pig (Gibbins et al. 1987). The combina- tion C G R P + tachykinin (substance P) + galanin has been previously described in the rat (Ju et al. 1987; Skofitsch and Jacobowitz 1985a, b), but there is no evidence at the

moment that it might correspond to a specific peripheral projection. The functional significance of peptide colocal- isation in dorsal root ganglion cells is still a matter of de- bate. The topic is particularly delicate since negative results are difficult to explain and positive results may reflect cross- reactivity to known or unknown antigens. The reported observations of Gibbins and colleagues (1987) suggest that combinations of certain peptides in sensory neurones may have tissue-specific projection pathways. An alternative viewpoint is that cells that convey different types of nocicep- tive sensory stimuli may contain different combinations of peptides. However, Leah and co-workers (1985b) were un- able to find a clear relationship between peptide content and sensory function in a combined electrophysiological and immunocytochemical study.

In conclusion, we have localised seven peptides in the spinal cord and dorsal root ganglia of the horse and pig by immunohistochemistry. The results obtained suggest that the overall pattern of distribution of peptide immuno- reactivities is in general agreement with that so far described in other mammals, although some species variations have been noted, particularly regarding the presence of immuno- reactive nerve cell bodies in different areas of the spinal cord.

Acknowledgements. This work was partially supported by grants of the Italian Consiglio Nazionale delle Ricerche and Ministero della Pubblica Istruzione. S. Kar is in receipt of a Commonwealth Scholarship. We wish to thank Drs. G. Terenghi for his critical revision of the manuscript, K.L. Valentino for the neurokinin A antiserum, G. Rindi for his valuable advice and suggestions and L. Bonfanti for technical help. The photographic expertise of Mrs. L. Chiappino is deeply appreciated.

References

Allen YS, Adrian TE, Allen JM, Tatemoto K, Crow T J, Bloom SR, Polak JM (1983) Neuropeptide Y distribution in the rat brain. Science 221 : 877-879

Anand P, Bloom SR (1984) Neuropeptides are selective markers of spinal cord autonomic pathways. TINS 7: 267-268

Anand P, Gibson SJ, McGregor GP, Blank MA, Ghatei MA, Bacarese-Hamilton AJ, Polak JM, Bloom SR (1983) A VIP- containing system concentrated in the lumbosacral region of human spinal cord. Nature 305:143-145

Anand P, Gibson SJ, Yiangou Y, Christofides ND, Polak JM, Bloom SR (1984) PHI-like immunoreactivity co-locates with the VIP-containing system in human lumbosacral spinal cord. Neurosci Lett 46:191-196

Basbaum AI (1985) A functional analysis of the cytochemistry of the spinal dorsal horn. In: Fields HL, Besson J-M (eds) Proceedings of the IV World Congress on Pain. Raven Press, New York, pp 121 145

Brown AG (1982) The dorsal horn of the spinal cord. Q J Exp Physiol 67:193-212

Bnrks TF, Cowan A, Porreca F (1985) Biological actions of bom- besin related peptides. Life Sci 37:105-153

Burnweit C, Forssmann WG (1979) Somatostatinergic nerves in the cervical spinal cord of the monkeys. Cell Tissue Res 200:83-90

Charnay Y, Paulin C, Chayvialle J-A, Dubois PM (1983) Distribu- tion of substance P-like immunoreactivity in the spinal cord and dorsal root ganglia of human foetus and infant. Neuros- cience 10:41-55

Cuello AC, Polak JM, Pearse AGE (1976) Substance P: a naturally occurring transmitter in human spinal cord. Lancet 2:1054~t056

Cuello AC, Del Fiacco M, Paxinos G (1978) The central and pe-

279

ripheral ends of the substance P-containing sensory neurons in the rat trigeminal system. Brain Res 152:499-510

Ch'ng JLC, Christofides ND, Anand P, Gibson S J, Allen YS, Su HC, Tatemoto K, Morrison JFB, Polak JM, Bloom SR (1985) Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal path- ways to injury. Neuroscience 16:343-354

Dalsgaard C-J (1988) The sensory system. In: Bj6rklund A, H6k- felt T, Owman C (eds) Handbook of chemical neuroanatomy, vol 6. Elsevier, Amsterdam, pp 59%636

Dalsgaard C-J, H6kfelt T, Johansson O, Elde R (1981) Somatosta- tin immunoreactive cell bodies in the dorsal horn and parasym- pathetic intermediolateral nucleus of the rat spinal cord. Neu- rosci Lett 27:335-339

Dalsgaard C-J, Heagerstrand A, Brodin E, Theodorsson-Norheim E, H6kfelt T (1985) Neurokinin A-like immunoreactivity in rat primary sensory neurons: coexistence with substance P. Histochemistry 83:407-414

Dalsgaard C-J, Jernbeck J, Stain W, Kjartansson J, Haegerstrand A, H6kfelt T, Brodin E, Cuello AC, Brown JC (1989) Calcito- nin gene-related peptide-like immunoreactivity in nerve fibres in the human skin: relation to fibres containing substance P-, somatostatin- and vasoactive intestinal polypeptide-like immu- noreactivity. Histochemistry 91:35-38

De Groat WC (1987) Neuropeptides in pelvic afferent pathways. Experientia 43 : 801-821

De Groat WC, Kawatani M, Hasamitsu T, Lowe I, Morgan C, Roppollo J, Booth AM, Nadelhaft I, Kuo D, Thor K (1983) The role of peptides in the sacral autonomic reflex pathways in the cat. J Auton Nerv Syst 7:339 350

Del Fiacco M, Cuello AC (1980) Substance P and enkephalin-con- taining neurons in the rat trigeminal system. Neuroscience 5:803-815

DiFiglia M, Aronin N, Leeman SE (1984) Ultrastructural Iocalisa- tion of immunoreactive neurotensin in monkey superficial dor- sal horn. J Comp Neurol 225 : 1-12

Fields HL, Emson PC, Leigh BK, Gilbert RFT, Iversen LL (1980) Multiple opiate receptor sites on primary afferent fibres. Nature 284:351-353

Fiocca R, Rindi G, Capella C, Grimelius L, Polak JM, Schwartz TW, Yanaihara N, Solcia E (1987) Glucagon, glicentin, proglu- cagon, PYY, PP and proPP-icosapeptide immunoreactivities of rectal carcinoid tumors and related non-tumor cells. Regul Pept 17 : 9-29

Forssmann WG (1978) A new somatostatinergic system in the mammalian spinal cord. Neurosci Lett 10:293-297

Fuxe K, Aganti LF, McDonald T, Locatelli V, H6kfelt T, Dals- gaard C-J, Battistini N, Yanaihara N, Mutt V, Cuello AC (1983) Immunohistochemical localisation of gastrin releasing peptide - bombesin-like immunoreactivity in the nervous sys- tem of the rat. Codistribution with substance P-like immunore- active nerve terminals and coexistence with substance P-immu- noreactivity in dorsal root ganglion cell bodies. Neurosci Lett 37:17-22

Gibbins IL, Furness JB, Costa M, Maclntyre I, Hillyard C J, Girgis S (1985) Co-localisation of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs. Neurosci Lett 57:125-130

Gibbins IL, Furness JB, Costa M (1987) Pathway-specific patterns of the coexistence of substance P, calcitonin gene-related pep- tide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea pig. Cell Tissue Res 248:417-437

Gibson S J, Polak JM (1986) Neurochemistry of the spinal cord. In: Polak JM, Van Noorden S (eds) Immunocytochemistry, modern methods and applications. J Wright and Sons, Bristol, pp 36C~390

Gibson S J, Polak JM, Bloom SR, Wall PD (1981) The distribution of nine peptides with special emphasis on the substantia gelatin- osa and on the area around the central canal (lamina X). J Comp Neurol 201 : 65-79

Gibson S J, Polak JM, Bloom SR, Sabate IM, Mulderry PM, Gha- tei MA, McGregor GP, Morrison JFB, Kelly JS, Evans RM, Rosenfeld MG (1984a) Calcitonin gene-related peptide immu- noreactivity in the spinal cord of man and of eight other species. J Neurosci 4:3101-3111

Gibson SJ, Polak JM, Anand P, Blank MA, Morrison JFB, Kelly JS, Bloom SR (1984b) The distribution and origin of VIP in the spinal cord of six mammalian species. Peptides 5 : 201-207

Gibson S J, Polak JM, Allen TE, Adrian TE, Kelly JS, Bloom SR (1984 c) The distribution and origin of a novel brain peptide, neuropeptide Y, in the spinal cord of several mammals. J Comp Neurol 227:78 91

Gibson S J, Polak JM, Anand P, Blank MA, Yiangou Y, Su H-C, Terenghi G, Katagiri T, Morrison JFB, Lumb BM, Inyama C, Bloom SR (1986) A VIP/PHI-containing pathway links uri- nary bladder and sacral spinal cord. Peptides 7:205-219

Goodman EC, Iversen LL (1986) Calcitonin gene-related peptide: novel neuropeptide. Life Sci 38:2169-2179

Harmann PA, Chung K, Briner RP, Westlund KN, Carton SM (1988) Calcitonin gene-related peptide (CGRP) in the human spinal cord: A light and electron microscopic analysis. J Comp Neurol 269:371 380

H6kfelt T, Efendic S, Hellerstr6m C, Johansson O, Luft R, Ari- mura A (1975a) Cellular localisation of somatostatin in endo- crine-like cells and neurons of the rat with special references to the AI cells of the pancreatic islets and to the hypothalamus. Acta Endocrinol 80 (Suppl) 200 : 5-41

H6kfelt T, Kellerth JO, Nilsson G, Pernow P (1975b) Substance P: localisation in the central nervous system and in some prima- ry sensory neurons. Science 190:889-890

H6kfelt T, Elde R, Johansson O, Luft R, Nilsson G, Arimura A (1976) Immunohistochemical evidence for separate popula- tion of somatostatin-containing and substance P-containing primary afferent neurons in the rat. Neuroscience 1:131-136

H6kfelt T, Ljungdfihl A, Terenius L, Elde R, Nilsson G (1977) Immunohistochemical analysis of peptide pathways possibly re- lated to pain and analgesia: enkephalin and substance P. Proc Natl Acad Sci USA 74:3081-3085

H6kfelt T, Schultzberg M, Lundberg JM, Fuxe K, Mutt V, Fah- renkrug J, Said SI (1982) Distribution of vasoactive intestinal polypeptide in the central and peripheral nervous systems as revealed by immunocytochemistry. In: Said SI (ed) Vasoactive intestinal polypeptide. Raven Press, New York, pp 65-90

Huang WM, Gibson S J, Facer P, Gu J, Polak JM (1983) Improved section adhesion for immunocytochemistry using high molecu- lar weight polymers of L-lysine as a slide coating. Histochem- istry 77 : 275-279

Hunt SP, Kelly JS, Emson PC, Kimmel JR, Miller R J, Wu J-Y (1981) An immunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superfical layer of the rat dorsal horn. Neuroscience 6:1883 1898

Jancs6 G, H6kfelt T, Lundberg JM, Kirfily E, Halasz N, Nilsson G, Terenius L, Rehfeld J, Steinbusch H, Verhofstad A, Elde R, Said S, Brown M (1981) Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and monoamine neurones using antisera to substance P, gastrin/ CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hy- droxytryptamine. J Neurocytol 10:963 980

Jessel T, Iversen LL (1977) Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature 268:549 551

Johansson O, H6kfelt T, Elde RP (1984) Immunohistochemical distribution of somatostatin-like immunoreactivity in the cen- tral nervous system of the adult rat. Neuroscience 13:265-339

Ju G, H6kfelt T, Brodin E, Fahrenkrug J, Fisher JA, Frey P, Elde RP, Brown JC (1987) Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, va- soactive intestinal polypeptide- and cholecystokinin-immunore- active ganglion cells. Cell Tissue Res 247:417-431

Kawatani M, Lowe IP, Nadelhaft I, Morgan C, De Groat WC

280

(1983) Vasoactive intestinal polypeptide in visceral afferent pathways to the sacral spinal cord of the cat. Neurosci Lett 42:311-316

Kawatani M, Nagel J, Houston MB, Eskay R, Lowe IP, De Groat WC (1984) Identification of leucine-enkephalin and other neu- ropeptides in pelvic and pudendal afferent pathways to the spi- nal cord of the cat. Soc Neurosci Abstr 10:589

Kernell D (1986) Organisation and properties of spinal motoneu- tones and motor units. Prog Brain Res 64:21-30

Lembeck F (1953) Zur Frage der zentralen Ubertragung afferenter Impulse. III. Mitteilung das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Rfickenmarks. Naunyn Schmiedebergs Arch Pharmacol 219:197-213

Leah JD, Cameron AA, Kelly WL, Snow PJ (1985a) Coexistence of peptide immunoreactivity in sensory neurons of the cat. Neu- roscience 16: 683-690

Leah JD, Cameron AA, Snow PJ (1985 b) Neuropeptides in physio- logically identified mammalian sensory neurones. Neurosci Lett 56: 257-263

Light AR, Perl ER (1979) Spinal termination of functionally identi- fied primary afferent neurons with slowly conducting myelin- ated fibres. J Comp Neurol 186:133-150

Ljungd/ihl A, H6kfelt T, Nilsson G (1978) Distribution of sub- stance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals. Neuroscience 3 : 861-943

Lundberg JM, H6kfelt T, Nilsson G, Terenius L, Rehefeld J, Elde R, Said S (1978) Peptide neurons in the vagus, splanchnic and sciatic nerves. Acta Physiol Seand 104:499-501

Lundberg JM, Terenius L, H6kfelt T, Tatemoto K (1984) Compar- ative immunohistochemical and biochemical analysis of pancre- atic polypeptide-like peptides with special reference to the pres- ence of neuropeptide Y in central and peripheral neurons. J Neurosci 4 : 2376-2386

Lundberg JM, Franco-Cereceda AF, Hua X, H6kfelt T, Fischer JA (1985) Co-existence of substance P and calcitonin gene-re- lated peptide-like immunoreactivities in sensory nerves in rela- tion to cardiovascular and bronchoconstrictor effects of capsai- cin. Eur J Pharmacol 108 : 315-319

Matthews MR, Cuello AC (1982) Substance P-immunoreactive pe- ripheral branches of sensory neurons innervate guinea pig sym- pathetic neurons. Proc Natl Acad Sci USA 79:1668-1672

McGregor GP, Gibson S J, Sabate IM, Blank MA, Christofides ND, Wall PD, Polak JM, Bloom SR (1984) Effect of peripheral nerve section and nerve crush on spinal cord neuropeptides in the rat; increased VIP and PHI in the dorsal horn. Neuros- cience 13:207-216

McNeill DL, Chung K, Carlton SM, Coggeshall RE (1988) Calci- tonin gene-related peptide immunostained axons provide evi- dence for fine primary afferent fibres in the dorsal and dorsolat- eral funiculi of the rat spinal cord. J Comp Neurol 207:303-313

Melander T, H6kfelt T, R6kaeus A, Tatemoto K, Mutt V (1984) Galanin immunoreactive neurons in the central and peripheral nervous system. Soc Neurosci Abstr 10:694

Molander C, Xu Q, Grant G (1984) The cytoarchitectonic organ- isation of the spinal cord in the rat. I. The lower thoracic and lumbosacral cord. J Comp Neurol 280:133-141

Nicoll RA, Schenker C, Leeman SE (1980) Substance P as a trans- mitter candidate. Ann Rev Neurosci 3 : 227-268

Panula P, Hadjiconstantinou M, Yang H-Y, Costa E (1983) Immu- nohistochemical localisation of bombesin/gastrin releasing pep- tide and substance P in primary sensory neurons. J Neurosci 3 : 2021-2029

Rexed B (1952) The cytoarchitectonic organisation of the spinal cord in the eat. J Comp Neurol 96:415-496

Rexed B (1954) A cytoarchitectonic atlas of the spinal cord in the cat. J Comp Neurol 100:297-379

R6kaeus A (1987) Galanin a newly isolated biologically active neu- ropeptide. TINS 10 : 158-164

R6kaeus A, Melander T, H6kfelt T, Lundberg JM, Tatemoto K, Carlquist M, Mutt V (1984) A galanin like peptide in the central nervous system and intestine of the rat. Neurosci Lett 47:161-166

Roppolo JR, Lowe IP, De Groat WC (1984) Immunocytochemical identification of leucine enkephalin in dorsal root ganglion cells of the rhesus monkey. Soc Neurosci Abstr I 0 : 993

Ruda MA, Coffield J, Bennett GJ, Dubner R (1983) Role of sero- tonin (5-HT) and enkephalin (ENK) in trigeminal and spinal pain pathways. J Dent Res 62:691

Ruda MA, Bennett GJ, Dubner R (1986) Neurochemistry and neural circuitry in the dorsal horn. Prog Brain Res 66:219-268

Salt TE, Hill RG (1983) Neurotransmitter candidates of somato- sensory primary afferent fibres. Neuroscience 10:1083-1103

Schroder HD (1984) Somatostatin in the caudal spinal cord: an immunohistochemical study of the spinal centers involved in the innervation of pelvic organs. J Comp Neurol 223:400- 414

Senba E, Shiosaka S, Hara Y, Inagaki S, Sakanaka M, Takatsuki K, Kawai Y, Tohyama M (1982) Ontogeny of the peptidergic system in the rat spinal cord: immunohistochemical analysis. J Comp Neurol 208 : 54-66

Seybold VS, Elde RP (1982) Neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat: I. Light micro- scopic studies of cell bodies and proximal dendrites. J Comp Neurol 205:89-100

Shu S, Ju G, Fan L (1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosei Lett 85:169-171

Skofitsch G, Jacobowitz DM (1985 a) Galanin-like immunoreactiv- ity in capsaicin sensitive sensory neurons and ganglia. Brain Res Bull 15:191-195

Skofitsch G, Jacobowitz DM (1985 b) Calcitonin gene-related pep- tide coexists with substance P in capsaicin sensory neurons and sensory ganglia of the rat. Peptides 6:747-754

Tracey DJ (1985) Ascending and descending pathways in the spinal cord. In: Paxinos G (ed) The Rat Nervous System. Academic Press, New York, pp 311-324

Tuchsherer MM, Seybold VS (1985) Immunohistochemical studies of substance P, cholecystokinin-octapeptide and somatostatin in dorsal root ganglia of the rat. Neuroscience 14:593 605

Valentino KL, Tatemoto K, Hunter J, Barchas JD (1986) Distribu- tion of neuropeptide K-immunoreactivity in the rat central ner- vous system. Peptides 7:1043-1059

Wanaka A, Matsuyama T, Yoneda S, Kimura K, Kamada T, Girgis S, Maclntyre I, Emson PC, Tohyama M (1986) Origins and distribution of calcitonin gene-related peptide-containing nerves in the wall of the cerebral arteries of the guinea pig with special reference to the coexistence with substance P. Brain Res 369:185-192

Wiesenfeld-Hallin Z, H6kfelt T, Lundberg JM, Forssmann WG, Reinecke M, Tschopp FA, Fischer JA (1984) lmmunoreactive calcitonin gene-related peptide and substance P coexist in sen- sory neurons to the spinal cord and interact in spinal behaviour- al responses of the rat. Neurosci Lett 52:199-204

Zieglg/insberger W, Tulloch IF (1979) The effects of methionine- enkephalin on spinal neurons of the cat. Brain Res 167:53-64