Substance P-containing projections in the dorsal columns of rats and cats

NeuroscienceVol.34, No. 3, pp. 607621, 1990 Printedin Great Britain

0306-4522/90$3.00+ 0.00 Pergamon Press plc 0 1990 IBRO

SUBSTANCE P-CONTAINING PROJECTIONS IN THE DORSAL COLUMNS OF RATS AND CATS F. CoNn,*t S. DE BIASI,$R. GIUFFRIDA$and A. RUSTIONI Departments

of Cell Biology and Anatomy, and Physiology, University Chapel Hill, NC 27599, U.S.A.

of North

Carolina

at Chapel Hill,

Abstract-Light and electron microscopic immunocytochemical methods were used to study the distribution and the morphology of substance P-positive fibers and axon terminals in the dorsal column nuclei of rats and cats, and to determine whether they are part of an ascending input to these nuclei. In rats, substance P-positive fibers and axon terminals are present throughout the rostrocaudal extent of the dorsal column nuclei. In cats, imrnunostained fibers and terminals are mostly confined to the ventral region of the caudal and middle portions of these nuclei but they are more homogeneously distributed at rostra1 levels. In both species, substance P-positive neurons are not present in the same nuclear complex. At the electron microscope level, substance P-positive terminals are small- to medium-sized and dome-shaped; they form asymmetric contacts on dendrites and contain many round, agranular vescicles and sparse dense core vescicles. In double-labeling experiments, visualization of substance P-immunoreactivity in the dorsal root ganglia and dorsal horn of the spinal cord was combined with the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase or of colloidal gold-labeled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase. These experiments show that substance P-positive axon terminals may originate from both small dorsal root ganglion neurons and from spinodorsal column nuclei neurons in lamina IV. Although quantitative evaluation of the contribution of these two pathways to the substance P innervation of the dorsal column nuclei has not been performed and other sources cannot be discarded on the basis of the present evidence, it is proposed that non-primary afferents to the dorsal column nuclei account for most of the substance P-positive fibers and terminals in the dorsal column nuclei. The experiments support previous findings suggesting that nociceptive input may access the dorsal column nuclei and that this may be mediated, though to a very limited extent, directly by way of small dorsal root ganglion neurons.

Substance P (SP) is one of the several tachykinins demonstrated in the mammalian CNS.28.35A role for SP in synaptic mechanisms is better established than for any other one of the tachykinins and its distribution in the CNS has been amply documented by means of radioimmunoassay S-45and immunocytochemistry. ‘5,26SP is released by dorsal root ganglion afferents to the spinal cord and its effects on dorsal horn neurons have been extensively studied.25.47,55 SP may be involved in neuronal communication at other levels of the somatosensory system since it is also present in neurons and/or fiber terminals in the

superficial laminae of the dorsal horn,3327the lateral cervical nucleuq2’ the trigeminal complex,‘4 the ventroposterior nucleus of the thalamus,5,40 and the somatosensory cortex. 29The selective distribution of SP-positive elements at all levels of the somatosensory system suggests an association with ascending, non-lemniscal, input mainly related to highthreshold, and, possibly, thermal receptors. High concentrations of SP in the brain were first reported in the nuclei gracile and cuneate’ (dorsal column nuclei; DCN). More recently, SP has been found in fibers in the fasciculi gracile and cuneate and in terminals in the corresponding nuclei.‘7.54We have undertaken this study to (1) define the distribution of the SP innervation within the DCN, (2) determine the ultrastructural features of SP-positive terminals and (3) verify that SP-positive fibers are part of the ascending spinal input to the DCN. Preliminary observations have been previously reported. 12.53

*To whom correspondence should be addressed. TPresent address: Institute of Human Physiology, University of Ancona, I-6013 1, Ancona, Italy. JPresent address: Department of General Physiology and Biochemistry, Section of Histology and Human Anatomy, University of Milan, I-20133, Italy. §Present address: Institute of Human Physiology, University of Catania, I-95125, Catania, Italy. Abbreviations : ABC, avidin-biotin complex; CUN, cuneate nucleus; DAB, 3,3’-diaminobenzidine; DCN, dorsal column nuclei: DRG, dorsal root ganglia; ECUN, external cuneate nucleus; GN, gracile nucleus; NGS, normal goat serum; PAP, peroxidase-antiperoxidase complex; PB, phosphate buffer; SP, substance P; TBS, Tris-buffered saline; WGAapoHRI-Au, wheat germ agglutinin apoHRP-gold; WGA-HRP, wheat germ agglutinin-conjugated horseradish peroxidase.

EXPERlMENTAL PROCEDURE5 Antisera Three different SP antisera were used. The first one, from Immunonuclear Co. (Stillwater, MN: lot no. 85444016), was generated in a rabbit against a keyhole limpet hemocyanin conjugate, and was used at 1: 3000 dilution. The second, kindly provided by Dr G. Nilaver, was raised in a 607

F. CONTIet al.

608

rabbit against synthetic SP conjugated to bovine thyroglobuline by carbodiimide; its specificity has been documented by preabsorption tests.’ This antiserum was used at 1: 4000 dilution. Most of the observations are from material processed with an antibody prepared and characterized in Dr Petrusz’s laboratory and used at I : 500&l : 10,000 dilution. Characterization of this antiserum is incomplete, but preliminary results show that it does not cross-react significantly with substance K. In the present paper, we use the term “SP-positive” to indicate immunoreactivity to these polyclonal antisera. The possibility that they may recognize other tachykinins, which have the same carboxyl terminal amino acids as SP, cannot be entirely ruled out. For both the light and electron microscopical experiments described below, controls included (i) omission of the primary antiserum, (ii) replacement of the primary antiserum by normal serum or buffer solution, (iii) incubation with diluted antiserum preabsorbed with an excess of antigen. In these cases, no specific staining was observed in the DCN and in the dorsal horn of the spinal cord. Immunocyrochemisrry dorsal column nuclei

of substance

P-innervation

of the

Lighr microscopy. Eight adult male Sprague-Dawley rats (3044382 in Table 1) and two Mongrel cats (from the University of North Carolina) (541 and 557 in Table 1) were used for this part of the study. Rats were anesthetized with chloral hydrate (40 mg/lOO g) and cats with sodium pentobarbital (30 mg/kg). Six of the eight rats and the two cats were perfused with physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.2-7.4. In the two other rats (310 and 330) 1 ~1 of a solution of colchicine in physiological saline (100 pg/pl) was injected in the fourth ventricle via a glass micropipette glued to a microsyringe 2448 h before perfusion. The brains were removed, postfixed overnight in the same fixative at 4°C and 25-pm-thick coronal sections were cut on a Vibratome from a block of the medulla containing the DCN. Sections were collected in 0.1 M PB in serial groups of three. One of the three sections was processed for immunocytochemistry; one was counterstained with 0. I % Cresyl Violet. Before immunostaining,

Table 1. Summary and details of the experimental protocols of the animals used for the light microscopic study of substance P-innervation of the dorsal column nuclei and of substance P-positive neurons in the spinal cord Rat 304 310 312 320 330 363 381 382 414 428 459 460 461 500 547

Colchicine

(IV

Immunocytochemistry spinal cord.

of substance P-positive neurons in

Before performing double-labeling experiments (see below) to verify the ascending origin of SP innervation of the DCN, the possibility that dorsal horn neurons may contain high levels of SP was tested in seven rats (414-547 in Table 1) and one cat (560 in Table 1). In three of the rats, colchicine (100 pg/pl in physiological saline) was injected, in doses between 0.5 and 1 pl by way of single or multiple injections, in the left dorsal horn of one to two spinal segments between C2 and C7. In four rats and in the cat. unilateral colchicine injections were made at levels caudal and very close to a transection of the left dorsal and dorsolateral funiculi at levels varying between C3 and C6. After 24 h, all animals were perfused as described above, the spinal cord was dissected, and 25-pm-thick sections were cut with a Vibratome. Sections were collected, processed for immunocytochemistry, and charted as described above for light microscopical material.

Suinal transection Double-labeling experinlenls

1.0 pl (IV v.)

1.0 n1 (IV v.)

0.1 0.1 0.1 2.1

/I1 /ll /I1 pl

left DF-DLF left DF-DLF left DF-DLF left DF-DLF

0.5 /I1 0.5 /I1

Cat 541 557 560

sections were pretreated with ethanol (50”, 70”. 50,‘, 15 min each) and rinsed in Tris-buffered saline (TBS). They were then incubated for 30 min in 10% normal goat serum (NGS) in TBS, for 16-20 h in the primary antiserum at 4°C and, after rinsing in TBS, were processed according to the peroxidase-antiperoxidase (PAP) method or to the avidin biotinylated peroxidase complex procedure (ABC, Vector). Immunostaining was then visualized by 3,3’-diaminobenzidine (DAB, Sigma; 0.075% in 0.05 M Tris buffer at pH 7.6) and 0.002% H,O, for 778 min. Following three rinses in TBS, sections were mounted on gelatin-coated slides, air-dried and coverslipped with DPX. The distribution of immunostained fibers and terminals was charted by means of a drawing tube using a 40 x objective. The architectonic boundaries of the DCN and adjacent structures were added by superimposing the corresponding Nissl-stained section upon the plotting of immunopositive material. Rostrocaudal levels were referenced to distance, in mm above (+) and below (-) obex. Elecfron microscopy. Five rats (not listed in Tables) were perfused with 4% paraformaldehyde and 0.551% glutaraldehyde in PB. After overnight post-fixation, the lower brainstem was cut with a Vibratome in 40-pm-thick sections that were processed for pre-embedding SP immunocytochemistry as described above, using the antiserum provided by Dr Petrusz. Sections were osmicated, dehydrated, and flat-embedded in Epon. Thin sections were mounted on formvar-coated grids and stained with uranyl acetate and lead citrate.

0.1 /I1

left DF-DLF

v. = fourth ventricle; DF = dorsal DLF = dorsolateral funiculus.)

funiculus;

In eight rats a retograde tracer was injected in the left cuneate nucleus (Table 2). Four rats received injections of wheat germ agglutininHRP (WGA-HRP, Sigma 2% in distilled water) and the remaining four rats received injections of colloidal gold-labeled WGA conjugated to enzymatically inactive HRP (WGAapoHRP-Au).4 After 36-72 h, all animals were re-anesthetized with pentobarbital (35 mg/kg), and a laminectomy was performed to expose the dorsal surface of the spinal cord. Except for one animal (794). colchicine (SO-100 pg/pl in physiological saline) was injected in doses between 0.2 and 0.8 ~1 in the left dorsal quadrant of one to two spinal cord segments between C2 and C7. In two of the rats (498 and 499) a transection of the dorsal and dorsolateral funiculus was made immediately rostra1 to the colchicine injection. In rat 794, a piece of aelfoam (6 x 3 mm wide) soaked with 10 ~1 of colchicine T50 pg/pl) was applied unilaterally onto the pial surface of the left dorsal column and dorsolateral funiculus at C5C7. Twenty-four to 32 h after colchicine administration, rats were re-anesthetized and perfused with physiological saline or 1% paraformaldehyde, followed by 4% paraformaldehyde in PB. The medulla and the spinal cord segments in

609

Substance P in the dorsal column nuclei Table 2. Summary and details of the experimental protocols of the rats used for the double-labeling of neurons in the dorsal ganglia and spinal cord Rat 489 498 499 504 771 175 776 194

Colchicine O.Spl 0.5 /II OSpl 0.5 pi 0.8 ~1 0.8 /11 0.8 ~1 gelfoam

Spinal transection

Retrograde tracer

Injections

left DF-DLF left DF-DLF

WGA-HRP (2%) WGA-HRP (2%) WGA-HRP (2%) WGA-HRP (2%) WGAapoHRP-Au WGAapoHRP-Au WGAapoHRP-Au WGAapoHRP-Au

0.3 /I 1 0.3pl 0.3 /I 1 0.5 p 1 0.8 ~1 0.25 /ll 0.4jll 0.6~1

-

-

which colchicine was applied, as well as segments above and below the application sites, were post-fixed in 4% paraformaldehyde for 1-2 days. Twenty-five-micron-thick sections were cut from these blocks with a Vibratome and collected in serial groups of five. In rats 771, 775, 776 and 794, cervical dorsal root ganglia (DRG) ipsilateral to the colchicine application were also dissected out; these were embedded in wax, and one 5-pm-thick section every 10th was mounted on subbed slides and air-dried. One Vibratome section out of each group and the paraffin sections were processed for the histochemical visualization of the retrograde tracer. In rats injected with WGA-HRP, a cobaltenhanced protocol which yields a granular black reaction product was used.6 WGAapoHRP-Au was revealed by silver intensi~~tion of gold particles (15 min in the dark and at room temperature; Intense II, Janssen). Sections of spinal cord segments and DRG were subsequently processed for SP immunocytochemistry as described above. RESULTS Substance P-annexation of the dorsal cohmn

nuclei

SP-positive fibers are present in the gracile and cuneate fasciculi of all rats studied (Figs 1 and 4A). Fibers are also in the peripheral regions of the DCN, and, more sparsely, in the central regions (Figs 1 and 4B). SP-positive terminal-like profiles (referred to, hereafter, as terminals) have a widespread distribution in the DCN. No obvious differences are observed in the distribution of SP-positive terminals along rostrocaudal levels of the nuclei. Immunostained terminals are present from the most caudal to the most rostra1 levels (Fig. 1). A higher density of positive terminals is present in the cuneate than in the gracile nucleus. In the regions adjacent to the DCN, terminals are dense in the trigeminal nuclear complex and in the nucleus of the solitary tract. Immunostaining in these structures is clearly stronger than in the DCN. No SP-positive perikarya are present in the DCN even in the two rats with injection of colchicine in the fourth ventricles; in these rats the intensity of fibers and terminals staining is lower than in untreated rats. The distribution of SP-immunoreactivity in the DCN of cats is different from that of rats. At caudal-most levels immunostaining is sparse and mostly confined to the ventral cuneate region (-5, Figs 2 and 4C). Proceeding rostrally SP-positive fibers and terminals in the cuneate nucleus (CUN) are almost exclusively present in the ventral region and along the margin of the nucleus; very few terminals

are present in the dorsal part of middle cuneate, i.e. from around obex to about 4 mm caudal to it.9 In the gracile nucleus (GN), the terminals are sparser than in the cuneate; however, a preferential localization of the reaction product is evident in its ventral part. Rostra1 to obex, the distribution of the reaction product in the DCN becomes gradually more diffuse and uniform (+ 1 and + 2, Fig. 3). At the most rostra1 levels examined in detail, immunostaining is present in the external cuneate nucleus (ECUN) and in a region corresponding to nucleus Z (+3.5, Fig. 3). Electron microscopic immun~yt~hemist~ reveals the morphology, cytological details, and synaptic relations of SP-positive terminals in rats (Fig.5). These are mostly small- to medium-sized, domeshaped terminals making asymmetric contacts on one, or occasionally more than one, small or large dendrite (Fig. SA and B). SP-positive terminals contain many round agranuiar vesicles of small size and only a few scattered dense core vesicles (Fig. 5C). Dendrites postsynaptic to SP-stained terminals are also contacted by unstained boutons containing a granular or flat vesicles (Fig. 5D). Close apposition between SP-stained and unstained terminals, suggestive of axo-axonic synapses, was observed although evidence of the direction of such synapses is elusive (Fig. 5B and D). Substance P-positive neurons in the spinal dorsal horn

In rats that received colchicine injections, immunoreactive neurons are numerous throughout the dorsal horn (Fig. 9A and B); their density decreases symmetrically on both sides rostrally or caudally from the injection site. SP-positive neurons in segments away from the injection site or in segments of untreated animals are primarily small neurons in lamina II. In the rat with only transection of the dorsal and dorsolateral funiculi, SP-positive neurons are numerous in all laminae of the dorsal horn at levels immediately below the lesion. The density of SP-positive neurons appears higher in cases in which the colchicine injection is combined with the transection of the dorsal quadrant. The relevant findings in rats and in the cat so treated are summarized in Fig. 6. In the segment immediately below the lesion at C4 in the rat and at C5-C6 in the cat, numerous neurons are present in the dorsal horn of both sides, even though they are denser on the treated side.

ECUN

CUN ,

GN

-0.7

Fig. 1.Camera lucida reconstruction of four sections from the DCN of rat 3 12. Dots and short wavy lines represent immunoreactive punctate materials and fibers, respectively. Open circles represent neurons plotted in the Nissl-counterstained adjacent section. The blackened region in the inset (coronal sections of the medulla oblongata) represents the region enlarged. Numbers indicate the distance from the obex in mm. Scale bar = 100 pm for the inset, and 100 pm for the enlarged regions. 610

Substance

Fig. 2. Camera

lucida reconstruction

P in the dorsal

column

of three sections from caudal 1. Scale bar = 200 pm.

nuclei

DCN of cat 557. Symbols

611

as for Fig.

F. CONTIet al.

612

-,L Fig. 3. Camera

lucida reconstructions

CUN of three sections from middle and rostra1 DCN of cat 557. Symbols as for Fig. 1.

Substance

P in the dorsal

column

nuclei

Fig. 4. (A) An SP-positive fiber in the gracile fasciculus of rat 3 12. (B) An SP-positive fiber in the central part of the cuneate nucleus of the same rat as in (A). (C) SP-positive terminals in the cuneate of cat 557; these are denser in the ventral region (bottom of figure) than in the dorsal region (top of figure) of the nucleus. (D) SP-positive fibers and terminals at the dorsal margin of the cuneate of the same cat as in C. Scale bar = 30pm for (A), and 15 pm for (B), (C) and (D).

613

Fig. 5. Electron micrographs of SP-positive terminals in the cuneate nucleus. These can hc cas~ly detected against the immunonegative background and may be more than one in a small field, conlxting dllferent dendrites (A) or the same one (B). lmmunopositive terminals may display dense core vcs~~~les 111the plane of the section and can be dome-shaped and surrounded by a thin glial sheet (C‘) or in CIOW apposition to another immunonegative ending (D). Scale bars = I Itrn and 0.5 I’m 614

Substance P in the dorsal column nuclei

615

Fig. 6. Camera lucida reconstruction of SP-positive neurons in the spinal cord of rat 414 (left column) and cat 560 (rig& column) which received colchicine injection and dorsal quadrant lesion. Each drawing is the result of su~~mposing five sections. Each dot is one neuron. SP-positive neurons are densest in lamina II and in laminae IV and V below and ipsilateral to the injection/lesion. SP-positive neurons are denser in lamina II, but they are also present in more ventral laminae. In the segments above the lesion, up to C2 in the rat and up to C4 in the cat, SP-positive neurons are present but less numerous than caudal to it (Pig. 6), and are mostly located in lamina II; scattered immunoreactive neurons are present in laminae III, IV, V and X. Double-libeling

experiments

In the early experiments of the group, it became apparent that (a) combined lesion and colchicine injection resulted in loss of retrograde labeling from dorsal horn neurons in sections with the most evident SP-positive cell bodies and (b) such “leakage” of retrograde labeling, though still occurring, was less prominent in animals treated only with colchicine. As a result, the best simultaneous visualization of retrogradely labeled and SP-positive dorsal horn neurons occurred in rats treated only with colchicine and in a short span of cord tissue immediately below and above the injection. It was also quite apparent that, for the purpose of double-ladling both in DRG and spinal dorsal horn, best results were obtained with the retrograde transport of WGAapoHRP-Au. For these reasons, the following description is based upon rats with medullary injections of WGAapoHRP-Au and colchicine treatment only.

In the DRG examined from these rats two major populations of labeled neurons are observed in sections where tracer visualization and immunocytochemistry are optimal: (a) neurons labeled by the retrograde transport of WGAapoHRP-Au; and (b) neurons stained only by SP immunocytochemistry. Neurons in (a) are, on the whole, larger that neurons in (b). A very small fraction of medium-sized neurons in these sections is labeled for both markers (Fig. 7). Their number varies according to the volumes of injected colchicine and is highest in rat 794, possibly as a result of the application of colchicine-soaked gelfoam directly onto the dorsal columns. Quantitative data from this rat are summarized in Table 3. In the spinal cord of rats 771, 775 and 776, retrogradely labeled neurons at levels above the colchicine injection are mostly in the medial part of the lamina IV of the dorsal horn ipsilateral to the injection site; they are also present in laminae III and V, but only sparse in deeper laminae. Closer to the colchicine injection site, retrogradely labeled neurons are still present but less numerous. Black granules indicative of retrograde transport tend to label cell processes rather than cell bodies. SP-positive neurons are numerous at levels through colchicine injection (Fig. 8 a-d, left column). Some also contain, in their cell body or processes, the black granular reaction

616

F. CONTIet al.

Fig. 7. Photomicrograph from C5 dorsal root ganglion of rat 794. Large cell bodies are for the most part labeled with dark grains of the silver-intensified retrograde tracer. SP-positive cells contain brown diffuse reaction product and are of small size. One SP-positive neuron also contains the grains of the silver-intensified retrograde tracer (arrow). Scale bar = 20 pm.

product typical of retrogradely labeled neurons. It is, however, difficult to determine whether such neurons are numerous because (1) optimal visualization of both markers is not always attained, and (2) in some neurons, the presence of silver-intensified gold particles tends to mask the immunostaining. Therefore, only few neurons are unequivocally double-labeled and these are present in lamina IV. Caudal to the injection site, SP-positive cell bodies disappear and numerous retrogradely labeled neurons are present; a situation similar to that at levels above the colchicine injection. In rat 794, in which colchicine was applied on the dorsal surface of the spinal cord, the gradient of labeling is similar to that in rats with colchicine injection (Fig. 8, right column). In this rat, only retrogradely labeled neurons are present in laminae III-V rostra1 to the application of colchicine (Fig. 8a’). In segments overlaid by the application of colchicine, SP-positive neurons are intermingled with neurons whose cell bodies and/or dendrites contain black grains indicative of the presence of retrogradely transported WGAapoHRP-Au (Fig. 8b’d’). Like in rats with colchicine injections, some neurons are clearly double-labeled in lamina IV (Fig. 9C), but in many cases this identification is uncertain. Caudal to the application of colchicine, labeling is as described in rats with colchicine injections.

DISCUSSION

SP has attracted much attention as a possible neuromediator released at the peripheral and central endings of unmyelinated (and myelinated) axons of DRG neurons.13.43 It is probable that this peptide is involved in the transmission of nociceptive and thermal information34 and that it is co-released with glutamate, I6 SP is probably also released by endings in the DCN: weak inhibitory effects on neurons of these nuclei were observed in an early study*’ but, later, slow excitatory effects were reported.“,‘* From the present results it appears that SP innervation of the DCN originates from both DRG and dorsal horn neurons. Though this conclusion is in apparent conflict with the lesion studies of Kojima and Kanazawa,” it is probable that, under normal circumstances, SP content in the DCN is too low to be significantly reduced after dorsal columns transection. A primary afferent origin of SP innervation of the DCN of cats was proposed on the basis of the similar ultrastructure of immunopositive endings and dorsal root afferent terminals.s4 In the present electron microscopial observations in rats, it is apparent that SP-positive terminals vary in their morphological features but at least some of them have characteristics associated with a primary afferent origin, e.g. round

Substance P in the dorsal column nuclei

Fig. 8. Camera lucida reconstruction of the results obtained in two double-labeling experiments; rat 775 (left column) which received colchicine in four penetrations in C3 and C4 (see inset), and rat 794 (right coIumn) in which a ~ctangular piece of gelfoam, soaked in colchicine, was applied over the dorsal surface of the spinal cord on the left side at CSC7 (see inset). In the drawings of the dorsal horn, SP-positive neurons are represented by open profiles, retrograde labeling in perikarya or dendrites is represented by fine dots, and double-labeled neurons are represented by filled profiles. Regularly patterned dot matrix represent the extent of the superficial laminae (I and II) of the dorsal horn.

vescicles, asymmetric contacts and apposition by small endings with pleomorphic vesicles. The existence of very sparse SF-positive neurons projecting to the DCN is in agreement with the limited overlap of the soma size of SP-positive4‘j and DCN-projecting neuronsz4 in the DRG. We have not tried to determine whether SP-positive primary afferents to the

DCN are unmyelinated, or whether these belong to the small proportion of thinly myelinated SP afferents.18 The latter is most probable since it remains to be established whether any of the large number of unmyelinated fibers in the dorsal columns3’ ascend to medullary levels. If indeed SP-positive primary afferents ascending to the DCN are thinly myelinated,

F. CONII et al.

618

Fig. 9. (A) SP-positive neurons in the cervical spinal cord of rat 460, at a level immediately below the colchicine injection site. (B) Higher magnification of SP-positive neurons in lamina IV of the dorsal horn of rat 460. (C) Photomicrograph taken at a level corresponding to the one shown in Fig. 8b’ showing one neuron labeled by black grains of WGAapoHRP-Au (left) and one SP-positive neuron (right) also labeled (arrows) by the retrograde transport of WGAapoHRP--Au in the DCN. Scale bars = 300 pm for (A); 80pm for (B); and 15 pm for (C).

they would most likely mediate activation of heatsensitive nociceptors and/or thermal (cooling) receptors. Whatever modaIity these fibers may be associated with, it is hard to speculate on their functional role considering their small number. It is probable that sensory information, however modest, along a given pathway may be of relevance in the operational scheme of parallel processing. The striking overlap of the distribution of SPpositive fibers and terminals and of non-primary afferents in the DCN of cats, where both systems are concentrated ventrally and rostrally and largely spare the dorsal part at middle levels of these nuclei,4g,49 suggests a spinal cord origin for probabIy the majority of the SP-innervation of these nuclei. SP-positive

terminals are homogeneously distributed in the DCN of rats, where non-primary afferents do not show topographical segregation.” The heterogeneous morphology, at the electron microscope, of stained terminals is compatible with an origin of SP-positive afferents to the DCN from the dorsal horn as well as from the DRG. A spinal origin for SP-positive afferents to the DCN is supported by the presence of SP-positive neurons in deep laminae of the dorsal horn where DCN-projecting neurons are located.12 Immunopositivity in these neurons in colchicinetreated animals probably results from a postulated role of colchicine in perikaryal concentration of substances that are normally transported along axons down to their terminals. It is unlikely that high levels

Table 3. Quantitative data on substance P-positive neurons (middle row of numbers) out of retrogradely labeled neurons (upper row of number) in the dorsal root ganglia (C4-Tl) of rat 794 c4

c5

615 9 1.5

344 5 1.5

Percentages

C6

C7

1104 12 1.1

are in bottom

1210 16 1.3

row of numbers.

C8 1408 14 1.0

Tl

Total

350 5 1.4

503 I 59 1.2

Substance

P

in the dorsal column nuclei

of SP in dorsal horn neurons result from indiscriminate up-regulation of this peptide since, although the drug must have diffused extensively (note bilateral immunostaining of cell bodies, Fig. 6), hardly any neuron in the ventral horn was immunopositive. That the effect of colchicine does not result in spurious immunostaining is supported by accumulation of SP in fibers and neurons that may contribute to ascending paths after spinal transection as shown here and in Furthermore, recent evidence previous studies. 37,41.44 from in situ hybridization has revealed that some neurons in lamina IV of rats contain mRNA for SP.” Double-labeling experiments provide further basis for the claim that at least some of the SP-positive dorsal horn neurons are at the origin of non-primary afferents to the DCN. This evidence should be considered in the light of two limitations. The first one stems from the difficulty in obtaining adequate infiltration of the DCN by the tracer without involving adjacent structures that may receive projections from the dorsal horn. However, lamina IV neurons are retrogradely labeled by injection of the dorsal medula that involve the DCN,22.50while infiltration by the tracer of neighboring nuclei, especially the solitary nucleus, results in labeling of neurons in laminae I, V and X.39 The second limitation consists in the partial perikaryal depletion of retrograde tracer as a result of the colchicine application. Perikaryal depletion was accompanied by visualization of longer segments of dendrites than are detectable under normal circumstances. This phenomenon may be due to a colchicine-induced dendritopetal transport of the tracer, operated by a mechanism probably similar or identical to that reported for lysosomal enzymes after colchicine treatment.23 As a result of this, the impression was gained that many more dorsal horn neurons may have been retrogradely labeled and

619

immunostained than were unequivocally doublelabeled. The presence of double-labeled neurons in a very limited thickness of cord tissue immediately adjacent to the colchicine injection may explain the failure to detect SP-containing neurons of origin of non-primary afferents to the DCN in a previous similar study. 33a We may conclude that SP innervation of the DCN have multiple origins and the ascending component is likely to be mainly from non-primary afferents. Since at least 50% of the neurons of origin of non-primary afferents respond to noxious stimuli, 2,30*42 it is conceivable that SP-containing spinomedullary neurons may account for most of the nociceptive responses recorded in the DCN (see Ref. 18 for data and literature), although a very limited contribution seems to arise also from DRG neurons. The question of which transmitter is released by the SP-positive synaptic endings remains to be established; in particular it will be worth exploring whether, like in the superficial laminae of the dorsal hom,16 SP in the DCN may be released by terminals which are probably glutamergic. The possibility that SP-positive fibers in the DCN originate from brainstem and cortex has not been addressed by the present experiments. Neurons projecting from the brainstem tegmentum to the DCN are exceedingly sparse when retrograde tracer injections are confined within the boundaries of the DCN.52 The possibility that SP might be present in corticofugal projection and that it can be visualized in perikarya mostly in colchicine-treated material ” may deserve further exploration. Acknowledgements -This work was supported by USPHS grant NS-12440. The authors gratefully acknowledge K. Phend for assistance with the histological work, and C. Fern for typing.

REFERENCES of substance P and 5-hydroxytryptamine 1. Amin A. H., Crawford T. B. B. and Gaddum J. H. (1954) The distribution in the central nervous system of the dog. J. Physiol. 126, 596618. D. (1975) The dorsal column system. II. Functional properties and bulbar relay of the post-synaptic fibers 2. Angaut-Petit of the cat’s fasciculus gracilis. Expl Brain Rex 22, 471493. P. M., Roberts E. and Leeman S. E. (1979) The origin, 3. Barber R. P., Vaughn J. E., Slemmon J. R., Salvaterra distribution and synaptic relationships of substance P axons in rat spinal cord. J. camp. Neural. 184, 331-352. 4. Basbaum A. I. and Menetrey D. (1987) Wheat germ agglutinin-apo HRP gold: a new retrograde tracer for light- and electron microscopic single- and double-label studies. J. camp. Neurol. 261,306318. fibers in the thalamus from ascending 5. Battaglia G., Spreafico R. and Rustioni A. (1988) Substance P-immunoreactive somatosensory pathways. In Cellular Thalamic Mechanisms (eds Bentivoglio M. and Spreafico R.), pp. 365-374. Elsevier, Amsterdam. and serotonin projections to the rodent nucleus raphe 6. Benz A. J. (1982) The sites of origin of brainstem neurotensin magnus. J. Neurosci. 2, 829-842. A., Nilaver G., Mulhem J. and Zimmerman E. A. (1982) Intraventricular capsaicin: 7. Bodner R. J., Kirchgessner alterations in analgesic responsivity without depletion of substance P. Neuroscience 7, 631638. 8. Brownstein M. J., Mroz E. A., Kiser J. S., Palkovits M. and Leeman S. E. (1976) Regional distribution of substance P in the brain of the rat. Brain Res. 116, 299-305. 9. Cheema S. S., Whitsel B. L. and Rustioni A. (1983) The corticocuneate pathway in the cat: relations among terminal distribution patterns., cytoarchitecture, and single .neurons functional properties. Somatosensory Res. 2, 169-205. dorsal column .nathwav _ of the rat. III. Distribution of ascending 10. Cliffer K. D. and Giesler G. J. Jr (1989) Postsvnaotic afferent fibers. J. Neurosci. 9, 314&3168. I 11. Conti F., Fabri M., Abdullah L. and Manzoni T. (1988) Some pyramidal neurons in the somatic sensory cortex of cats contain tachykinin immunoreactivity. Sot. Neurosci. Abslr. 14, 717.

620

F. CON-I-1et

ai.

12. Conti F. and Rustioni A. (1986) Distribution of substance P-positive fibers and terminals in the dorsal column nuclei of rats and monkeys. Anat. Rec. 214, 25526A. 13. Cue110 A. C. (1987) Peptides as neuromodulators in primary sensory neurons. Neuropharmacology 26, 971-979. 14. Cue110 A. C., Del Fiacco M. and Paxinos G. (1978) The central and peripheral ends of the substance P-containing sensory neurons in the rat trigeminal system. Brain Res. 152, 499-510. 15. Cuello A. C. and Kanazawa I. (1978) The distribution of substance P immunoreactive fibers in the rat central nervous system. J. camp. Neurol. 17Z3, 12991.56. 16. De Biasi S. and Rustioni A. (1988) Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of the spinal cord. Proc. narn. Acad. Sci. U.S.A. 85, 7820-7824. 17. Del Fiacco M., Dessi M. L., Atzori M. G. and Levanti M. C. (1983) Substance P in the human brainstem. Preliminary results of its immunohistochemical localization. Brain Res. 264, 142-147. 18. Ferrington D. C., Downie J. W. and Willis W. D. Jr (1988) Primate nucleus gracilis neurons: responses to innocuous and noxious stimuli. J. Neurophysioi. 59, 886907. 19. Fox S., Krnjevic K., Morris M. E., Puil E. and Werman R. (1978) Action of baclofen on mammalian synaptic transmission. Neuroscience 3, 495-5 15. 20. Galindo A., Krnjevic K. and Schwartz S. (1967) Microiontophoretic studies on neurons in the cuneate nucleus. J. Physiol. 192, 350-377. 21. Giesler G. J. Jr and Elde R. P. (1985) Immunocytochemical studies on the peptidergic content of fibers and terminals within the lateral spinal and lateral cervical nuclei. J. Neurosci. 5, 1833-1841. 22. Giesler G. J. Jr, Nahin R. L. and Madsen A, M. (1984) Postsynaptic dorsal column pathway of the rat. I. Anatomical studies. J. ~euro~hysioi. 51, 260-275. 23. Gorenstein C., Bundman M. C., Lew P. J., Olds J. L. and Ribak C. E. (1985) Dendritic transport. I. Colchicine stimulates the transport of lysosomal enzymes from cell bodies to dendrites. J. Neurosci. 5, 2009-2017. 24. Gross H. H., Fox J. A. and Curtis J. D. (1979) A horseradish peroxidase study of primary afferent projections to the medullary cuneate nucleus in the rat. Neurosci. Lerl. 14, 147-152. 25. Henry J. L. (1976) Effects of substance P on functionally identified untis in cat spinal cord. Brain Res. 114, 4399452. 26. Hiikfelt T., Kellerth J. O., Nilsson G. and Pernow B. (1975) Substance P: localization in the central nervous system and in some primary sensory neurons. Science 190, 889-890. 27. Hunt S. P., Kelly J. S., Emson P. C., Kimmel J. R., Miller R. J. and Wu J. Y. (1981) An immunohistochemical study of neuronal populations containing neuropeptides or ~-aminobuty~te within the superficial layers of the rat dorsal horn. Neuroscience 6, 188331898. 28. Jesse11 T. M. and Womack H. D. (1985) Substance P and the novel mammalian tachykinins: a diversity of receptors and cellular actions. Trends Neurosci. 8, 43345. 29. Jones E. G., DeFelipe J., Hendry, S. H. C. and Maggio J. E. (1988) A study of tachykinin-immunoreactive neurons in monkey cerebral cortex. J. Neurosci. 8, 12061224. 30. Kamogawa H. and Bennett G. J. (1986) Dorsal column postsynaptic neurons in the cat are excited by myelinated nociceptors. Bruin Res. 364, 386390. 3 I. Kojima N. and Kanazawa I. (1987) Possible neurotransmitters of the dorsal column afferents: effects of dorsal column transection in the cat. Neuroscience 23, 263-274. 32. Krnjevic K. and Morris M. E. (1974) An excitatory action of substance P on cuneate neurons. Can J. Physiol. Pharmac. 52, 736744. 33. Langford L. A. and Coggeshall R. E. (1981) Unmyelinated axons in the posterior funiculi. Science 211, 176-177. 33a. Leah J. D., Menetrey D. and dePommery J. (1988) Neuropeptides in long ascending spinal tract cells in the rat: evidence for parallel processing of ascending information. ~eurosc~enee 24, 195-207. and biochemical correlations. Trends ~ei~ro~ri. 7, 34. Lynn B. and Hunt S. P. (1984) Afferent C-fibers: physiologi~l 186-188. A. Rec. Neurosci. 11, 13-28. 35. Maggio J. E. (1988) Tachykinins. of kassinin-like immunoreactivitv in rat central and 36. Maeeio J. E. and Hunter J. C. (1984) Regional distribution perivpYhera1 tissue and the effect of‘capsaicinr Brain Res. 307, 370-373. T. L. (1983) Distribution and origin of 37. Massari V. J., Tibazi Y. Park C. H., Moody T., Helke C. J. and O’Donohue bombesin, substance P, and somatostatin in cat spinal cord. Peptides 4, 673-681. P. W. and Lawson S. N. (1989) Cell type and conduction velocity of rat primary sensory neurons with 38. McCarthy substance P-like immunoreactivity. Neuroscience 28, 745-753. 39. Menetrey D. and Basbaum A. I. (1987) Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viscerovisceral reflex activation. J. camp. Neurol. 255, 439450. of certain neuropeptides in the primate thalamus. 40. Molinari M., Hendry S. H. C. and Jones E. G. (1987) Distribution Brain Res. 426, 270-289. S. J., Paul H. M. S., Lowman E. W. and Schlosser W. (1978) Localization and changes of 41. Naftchi N. E., Abrahams substance P in spinal cord of paraplegic cats. Brain Res. 153, 507.-513. excitatory and inhibitor input to neurons of the postsynaptic dorsal 42. Noble R. and Riddell J. S. (1988) Cutaneous column system in the cat. J. Physioi. 396, 497-5 13. M., Tsunoo A. and Akagi H. (1982) Role of substance P as a sensory transmitter 43. Otsuka M., Konishi S., Yanagisawa in spinal cord and sympathetic ganglia. In Subsfance P in the Nervous System (eds Porter R. and O’Connor M.), pp. 13-36. Pitman, London. 44. Pickel V. M., Miller R., Chan J. and Sumal K. K. (1983) Substance P and enkephalin in transected axons of medulla and spinal cord. Regul. Pept. 6, 121-125. for substance P. Narzcre 45. Powell D., Leeman S. E., Tregear G. W., Nial H. D. and Potts J. T. (1973) Radioimmunoassay New Biof. 241, 252-254. and quantitative examination of dorsal root ganglion neuronal sub46. Price J. (1985) An immunohistochemical populations. J. Neurosci. 5, 2051-2059. 47. Randic M. and Miletic V. (1977) Effects of substance P in cat dorsal horn activated by noxious stimuli. Bruin Res. 128, 1644169. afferents to the nucleus gracilis from the lumbar cord of the cat. Brain Res. Sl,81-95. 48. Rustioni A. (1973) Non-primary

Substance

P in the dorsal

column

nuclei

621

49. Rustioni A. (1974) Non-primary afferents to the cuneate nucleus in the bra&al dorsal funiculus of the cat. Bruin Res. 15, 247-259. 50. Rustioni A. and Kaufman A. B. (1977) Identification of cells of origin of non-primary afferents of the dorsal column nuclei of the cat. Expl Brain Res. 17, I-14. 51. Warden M. K. and Young W. S. III (1988) Distribution of cells containing mRNAs encoding substance P and neurokinin B in the rat central nervous system. J. camp. Neural. 272, %113. 52. Weinberg R. J. and Rustioni A. (1989) Brainstem connections to the rat cuneate nucleus. J. camp. Neural. 282, 142-156. 53. Wen C. Y., De Biasi S., Van Eyck S. L., Petrusz P. and Rustioni A. (1987) Substance P terminals in rat cuneate nucleus. Sot. Neurosci. Abstr. 13, 394. 54. Westman J., Blomqvist A., Kohler C. and Wu J. Y. (1984) Light and electron microscopic localization of glutamic acid decarboxylase and substance P in the dorsal column nuclei of the cat. Neurosci. ht. 51, 347-352. 55. Zieglgansberger W. and Tulloch I. F. (1979) Effects of substance P on neurons in the dorsal horn of the spinal cord of the cat. Brain Res. 166, 273-282. (Accepted 2 August 1989)