A spinomedullary projection terminating in the dorsal reticular nucleus of the rat

0306-4522~~$3.00+ 0.00 Pergamon Press plc 6 1990IBRO

~e~~~~~j~~re Vol. 34. No. 3, pp. 577-589, 1990 Printed in GreatBritain

A SPINO~EDULLARY PROJECTION TERMINATING IN THE DORSAL RETICULAR NUCLEUS OF THE RAT D. LIMA Institute of Histology and Embryology of the Faculty of Medicine and Centre of Experimental Morphology of the University of Oporto, 4200 Porto, Portugal Abstract-Spinal afferents to the medullary dorsal reticular nucleus were studied using the following retrograde tracers: horseradish peroxidase (diluted in dimethylsulfoxide), wheat germ agglutinin conjugated with horseradish peroxidase, and cholera toxin subunit B. Spinal cord cells projecting to that medullary region were located predominantly in medial lamina I and lamina X. Cell labelling was moderate in the medial part of laminae II-IV and sparse throughout laminae V-VII. Labelling was predominantly ipsilateral in the dorsal horn and bilateral in laminae VII and X. After mechanical lesions of the dorsal white matter which severed most of the ipsilateral cuneate fasciculus, the numbers of superficial dorsal horn cells that were labelled from the dorsal reticular nucleus were considerably decreased caudal to the lesion, which suggests that their axons utilize mostly the cuneate fasciculus. Since the medullary dorsal reticular nucleus of the rat has a predominant population of nociceptive specific neurons, it is suggested that this spino-dorsomedullary reticular pathway is involved in pain

PAG link possible by directly triggering the NRM descending inhibition. species by retrograde tracing techniques.‘,7s24.42*56 In contrast with the ventromedial and lateral reticInjection sites in the ventromedial reticular nuclei ular nuclei, the medullary dorsal reticular nucleus labelled spinal cord cells located in the dorsal horn (DRt) has not been shown to be a spinal target by neck and the ventral horn,‘.’ whereas those in the labelling methods. However, a recent study using the lateral reticular nucleus (LRN) labelled the same technique of antidromic activation has suggested that spinal areas,42*s6as well as the marginal zone.42 Spinal spinomesencephalic axons give collaterals to this part input to one of the ventromedial nuclei, the nucleus of the medullary reticular formation.37 On the other raphe magnus (NRM), has been confirmed in the hand, the DRt is known to contain neurons exclucat with electrophysiological techniques6 Such prosively or preferentially activated by noxious stimuli jections may be implicated in the transmission of from skin and viscera.@’ the spinal input to the higher centres, since the It is the purpose of the present work to investigate nucleus gigantocellularis of the ventromedial group the occurrence of spinal neurons projecting to the projects to the central median thalamic nucleus in the medullary dorsal reticular nucleus. The funicular rat.47 Spinomedullary connections may also play a trajectory of the ascending axons was also investimodulatory role, related to the functioning of the gated. The morphological features of the marginal spinal-midbrain pain modulatory 10op.~ This loop cells involved in this projection are described in is composed of spinomesencephalic axons exciting another article.32 Part of these results have been the periaqueductal gray (PAG), PAG neurons propresented in abstract form.29 cessing that information and activating the NRM, and the descending NRM axons which inhibit the EXPERIMENTAL PROCEDURES dorsal horn cells! However, the existence of a Fifteen male adult rats, 270-320 g in weight, were spinal-NRM projection6 may make a bypass of that anaesthetized intraperitonealiy with 35% chloral hydrate, 1ml/kg body weight. Animals were pressure injected in the Spinal projections to the reticular formation of the medulla oblongata have been revealed in several

CTb, cholera toxin subunit 8; DAB, diaminobenzidine; DCN, dorsal column nuclei; DF, dorsal funiculus; DLF, dorsolateral funiculus; DMSO, dimethylsulfoxide; DRt, medullary dorsal reticular nucleus; HRP, horseradish peroxidase; LRN, lateral reticular nucleus; LSN, lateral spinal nucleus; LT, tract of Lissauer; NRM, nucleus raphe magnus; PAG, periaqueductal gray; PBS, phosphate-buffered saline; Sol, nucleus of the tractus solitarius; SOIL, nucleus of the tractus solitarius, lateral; TMB, tetramethylbenzidine; VLF, ventrolateral funiculus; WGA-HRP, wheat germ agglutinin conjugated with horseradish peroxidase.

Abbreviations:

DRt with either 30% horseradish peroxidase (HRP) in 2% dimethylsulfoxide (DMSO), 2% wheat germ agglutinin coniugated with HRP (WGA-HRP). or 1.5% cholera toxin sub&& B (CTb). The i;jection v&&es are given in Table 1. The animals were positioned in a small animal stereotaxic frame, model 900 (David Kopff Instruments). Solutions were injected with Hamilton syringes held in the attached electrode manipulator, model 960. The syringes were driven by the manipulator and the plungers were driven by hand. The tracer was injected discontinuously, in small fractions, in order to make the injections last 5-15 min, according to tracer volumes. One to three injections were made in rostrocaudal sequence, as shown in Table 1. The stereotaxic

D. LIMA

578 Table RC -4.3 -4.3 -4.3;

Injection

site

DRt + DCN + SolL -4.8*

1. Summary

Tracer HRP/DMSO WGA-HRP CTb

of experimental

procedures

Total volumes injected (~1)

Experiments (no.)

0.16 0.16 3.20

224,248,25 151 503,565,566

Plane of sections

1

639 905,963,964 -4.3 -4.3;

-4.8*

DRt

-4.3;

-4.8;

WGA-HRP CTb

-5.3t

RC, distance in millimetres from the interaural *Two injections in rostrocaudal sequence. tThree injections in rostrocaudal sequence.

0.16 2.0

150 919

3.0

895 900

line to the centre of each injection,

parameters of Paxinos and Watson46 were followed. The rostrocaudal levels at which each injection was made are indicated in Table 1. In the first 11 animals in which the needle was kept perpendicular to the horizontal stereotaxic plane, the injection sites extended dorsally through the needle track to include the gracile and cuneate nuclei and the lateral part of the nucleus of the tractus solitarius (SolL) (Fig. IA-D; Table 1). To avoid involvement of the dorsal column nuclei (DCN) and SolL, another four animals were injected with the needle inserted obliquely, 29” lateral to the dorsoventral axis. The angular setting was adjusted in the protractor of the manipulator. The lateromedial distance from the sagittal plane at which the needle should traverse the pia-mater and the extent of its penetration in the nervous parenchyma to reach the DRt were calculated from the stereotaxic reference grid of Paxinos and Watson.46 Needles were left in situ for 15 min and then slowly retracted. Forty-eight hours later the animals injected with free HRP or WGA-HRP were re-anaesthetized as before and perfused through the ascending aorta with 5OOml saline followed by 1OOOml of 1.25% glutaraldehyde and 1% paraformaldehyde in phosphate buffer 0.12 M, pH 7.4-7.6.2’ The brain and the spinal cord were removed and immersed in the same fixative for 2 h and in 30% sucrose in phosphate buffer overnight at 4°C. The animals which received CTb were re-anaesthetized 72 h later and perfused with 4% paraformaldehyde in phosphate buffer 0.12 M, pH 7.4-7.6 followed by immersion of the pieces in the fixative for 2 h and in phosphate-buffered saline (PBS) overnight. Sections were cut at 50 pm in a freezing microtome in the case of the animals injected with free HRP or WGA-HRP, and in an Oxford Vibratome in those animals injected with CTb, at IOOpm through the injection sites, and at 75pm through the spinal cord. Coronal sections were cut through the injection sites and several spinal cord segments (Table I). Serial parasagittal sections of the entire mediolateral extent of the superficial dorsal horn were cut from some animals (Table 1). All of the coronal sections from each segment and

Abbreviations 10 12 V-VIP V, AP cu cu DF

dorsal motor nucleus of the vagus hypoglossal nucleus parvocellular portion of laminae V-VI lateral reticulated portion of lamina V area prostrema cuneate fasciculus cuneate nucleus dorsal funiculus dorsal root

at the rostrocaudal

coronal coronal parasagittal coronal parasagittal coronal parasagittal coronal coronal parasagittal coronal parasagittal parasagittal axis, according

Spinal segments examined

C,&, T2, T,, -I-,,-L, C,&,Tz>T,,T,,-L,

to Paxinos

and Watson.“”

all parasagittal sections were examined. Free HRP and WGA-HRP were revealed, respectively, by 3,3’-diaminobenzidine tetrahydrochloride (DAB) following the method of ltoh e/ uI.,‘~ and tetramethylbenzidine (TMB) according to De Olmos et a1.9 CTb was localized with the immunoperoxidase technique using two monoclonal antibodies (CTb, and CTb,, gift of Dr Marianne Wilkstriim, Gotebarg University), according to Erickson and Blomqvist.” The injection sites were drawn on cytoarchitectonic diagrams of the medulla oblongata taken from the atlas of Paxinos and Watson.46 The locations of the labelled cells in the spinal cord were recorded on diagrams of spinal cord segments showing Rexed’s laminae.48 These diagrams were constructed from coronal sections stained with Neutral Red. All cells from each spinal segment were counted. Cells which appeared to be only partly included in the sections were not considered. In three additional animals (nos 916. 946 and 965), the dorsal surface of the third cervical segment was exposed by laminectomy. The dorsal funiculus (DF) was cut superficially between the dorsal root entry zone and the midline using a sharp dissecting knife (Fig. 5AZ). CTb was then perpendicularly injected into the ipsilateral DRt. The injection sites covered the DRt, part of the DCN and the SOIL as in the 11 non-operated animals injected in the same way which are represented in Table 1. After 72 h the rats were perfused. Vibratome coronal sections were cut at 100pm through the injection sites. Seventy-five micrometre coronal sections were cut through the spinal cord C,-segment, and 75 pm parasagittal serial sections through segments C,-C, and C,C,, on both sides. Cells labelled in laminae 1, 11-111, IV-VI, VII and X were counted on the parasagittal sections rostrally and caudally to the spinal lesions. These numbers were compared with those obtained from three, non-lesioned control animals (nos 905, 963, 964) with similar injection sites (Table 1). To establish the limits of the tract of Lissauer (LT) at the level of the lesions, semithin Epon sections of the segment C, of two normal rats were

used in figures: DRt ECU gr Gr LT Sol spsc sp51

medullary dorsal reticular nucleus external cuneate nucleus gracile fasciculus gracile nucleus tract of Lissauer nucleus of the solitary tract nucleus of the spinal trigeminal nerve, caudal nucleus of the spinal trigeminal nerve, interpolar

Distribution

\

of spino-dorsomedullary

..r

*“’

projecting

neurons

519

,_

i .

*

Fig. 1. Examples of injection sites in coronal section. In A and B after a perpendicular of free HRP (no. 251). The inner halo in the micrograph (Fig. 1B) is outlined. The indicate the total area limited by that dashed lined at increasing distances beyond expressed in millimetres. In C and D after two perpendicular injections of CTb at a 964). The outlined area in D circumscribes the injection site which is represented in two oblique injections of CTb at a total of 2.0 ~1 (no. 919).

injection of 0.16 ~1 diagrams (Fig. 1A) the interaural line6 total of 3.20 ~1 (no. C. In E and F after

D. LIMA

580

stained with silver aminocarbonate.” The LT was recognized by having only small myelinated fibres in contrast with the dorsal funiculus (DF) and the dorsolateral funiculus (DLF) in which thick myelinated fibres occurred. The LT extended from the lateral curved tip of the dorsal horn to the cord surface with the dorsal limit passing immediately ventral to the entry of the dorsal root.

RESULTS Injection

sites

With both HRP tracers, injection sites presented a very dark central core and a surrounding zone in which extensions of the dense reaction alternated with lighter zones where no perikarya or nerve fibres were stained (Fig. 1B). This inner halo was considered as part of the injection site and is included in the corresponding drawings (Fig. IA). A fainter outer halo (Fig. 1B) in which labelled nervous elements occurred was excluded from the injection site because such cellular labelling is likely to result from uptake occurring in the inner zone.43 The injection sites of CTb were smaller than those obtained with the other tracers (Fig. IC-F), despite the much larger volumes injected. The compact core intermingled, at the periphery, with lighter patches in which no labelled neurons occurred (Fig. lD, F). This peripheral zone was therefore included in the injection site (Fig. lC, E). There was a diffuse labelling of the brain surface (Fig. lD, F) extending to the spinal cord (Fig. 3C, E), and of the area prostrema (Fig. ID, F). Cell groups lahelled in the spinal cord The spinal cord groups containing retrogradely labelled neurons after injection sites comprising the DRt, the DCN and the SOIL were located in laminae I, III-VI, VII and X, whatever the tracer used (Fig. 2A-C). The numbers of labelled cells per spinal area were larger with WGA-HRP (Fig. 2B) than with free HRP (Fig. 2A), and practically no cells occurred on the contralateral side (Fig. 2A, B). CTb injections determined considerably higher numbers of labelled cells in the ipsilateral and contralateral spinal cord (Figs 2C-E, 3A). There was a particularly intense ipsilateral labelling in medial lamina I and laminae IV-VI (Figs 2C-E; 3A, C, D; Table 2). In the cervical and lumbar segments examined lamina IV cells were concentrated in two separate groups located laterally and medially (Figs 2C, E, 3A, C). The very dense medial cell concentration extended to the medial, parvocellular part of laminae V-VI (Figs 2C, E, 3A). Labelled cells occurred bilaterally in laminae VII and X (Figs 2C-E, 3A; Table 2). Lamina III was moderately labelled on the ipsilateral side (Fig. 3A). Injections restricted to the DRt produced marked retrograde labelling in medial lamina I (Fig. 3B; Table 2) which was similar in intensity to that resulting from the larger injection sites (Fig. 3A; Table 2). Laminae IV-VI and VII were

scarcely labelled (Fig. 38, E, F; Table 2), whereas lamina X maintained a considerable amount of labelling (Fig. 3B; Table 2). Stained cells again occurred predominantly on the ipsilateral side, except in laminae VII and X in which cells were equally distributed through both sides (Fig. 3B; Table 2). The distribution of labelled cells along the spinal cord rostrocaudal extent depended on the tracer used irrespective of the injection site. After the two HRP markers labelling was high at C,-C, and it decreased caudally being practically null below C, in all spinal groups except lamina IV. Following CTb injections, labelling occurred at all spinal segments studied, but cell numbers were lower at the thoracic and lumbar cord (Fig. 2D, E) than at the cervical cord (Fig. 2C). At caudal levels the cell concentration in the medial part of laminae V-VI was less marked (Fig. 2D, E). General

morphology

qf’ labelled

cells

Retrograde labelling with free or conjugated HRP produced a granular precipitate in neuronal perikarya and first-order dendrites (Fig. 4A, D, E, H, I, L). At C,-C2 free HRP labelled more distal branches and some dendritic spines (see Ref. 32). The reaction product of CTb distributed diffusely in the cell bodies and the dendritic arbors up to thirdor fourth-order dendritic branches (Fig. 4B, C, F, G, J, K). A few HRP or CTb labelled neurons presented a thin, unbranched process which was interpreted as a presumable axon. Some lamina 1 cells possessed one such process abutting the dorsal funiculus (Fig. 4B). In coronal sections, after either large or small injections lamina I cells were roundish (Fig. 4A, B) with some ventrally directed primary dendrites which entered lamina II (Fig. 4B), or flat. elongated lateromedially (Fig. 4B) (see Ref. 32 for a detailed description). Lamina II cells were predominantly located along the dorsal border of the lamina (Fig. 4C). They presented roundish cell bodies with ventrally directed dendrites (Fig. 4C) which resembled the lamina I roundish cells. Lamina III cells were mainly fusiform with perikarya and primary dendrites oriented dorsoventrally (Fig. 4D). Lamina IV labelled cells presented either ovoid (Fig. 4E, F), or triangular somata (Fig. 4F). In the medial, parvocellular portion of laminae V-VI the majority of labelled cells was fusiform (Fig. 4G, H) with the perikarya and primary dendrites oriented parallel to the medial contour of the neck of the dorsal horn (Fig. 4G). Other cells presented roundish perikarya from which dendrites emanated in any direction (Fig. 4G). Most cells in lamina VII were triangular (Fig. 41, J). Their primary dendrites arose from the cell body angles (Fig. 41, J) and could be followed for rather long distances (Fig. 45). Lamina X cells were fusiform or ovoid and their dendrites variably oriented (Fig. 4K, L). In parasagittal sections, most cells had configurations

Distribution of spino-dorsomedullary

projecting neurons

581

Fig. 2. Distribution of spinal cord labelled cells in three experiments. (A) After free HRP (no. 251), (B) after WGA-HRP (no. 151) and (C-E) after CTb injections in the DRt + DCN + SolL (no. 639). Each diagram was constructed from 15 consecutive coronal sections from each one of segments C, (AC), T, (D) and L4(E). Dashed lines separate Rexed’s laminae which are indicated by roman numbers. Left side is ipsilateral.

similar to those appearing in coronal sections. Most lamina I cells were, however, elongated longitudinally

(Fig. DRt, cells (Fig.

3D, F). Following injections comprising the the DCN and the SolL some large triangular appeared in the lateral portion of lamina V 3C, D).

The attempts to sever the dorsal funiculi in three animals (nos 916, 946, 965) resulted in sectioning of the largest part of the euneate fasciculus and the medial part of the dorsal horn (Fig. 5A-C). The dorsal roots and the LT were spared (Fig. 5A-C). Although the configuration of the LT was markedly changed on the operated side (Fig. 5A-C), it could be

verified that the lesions did not invade the dorsal boundary of the LT. This boundary is a line joining the point at which the dorsal horn edge curves to reach its lateral tip, with the ventral contour of the dorsal root (Fig. 5A). In all cases the numbers of cells labelled rostrally to the spinal lesion were similar to those occurring in control animals (Fig. 51); Table 3). Caudally, those numbers were strongly decreased in all laminae but VII and X (Fig. SE; Table 3). In experiment 946 in which the lesion was more extensive, cell numbers at the ipsilateral side fell to nil in laminae I-III and were very low in laminae IV-VI (Table 3). Labelling on the contralateral side was less diminished. It amounted to about 10% of that in controis (Table 3).

582

D. LIMA

Fig. 3. (A and B) Diagrammatic representation of the distribution of cells retrogradely labelled from two experiments of CTb, obtained in each case from 15 consecutive coronal sections of segment C, In A (no. 964) the injection site encompassed the DRt + DCN + SolL. In B (no. 919) it was restricted to the DRt. Left side is ipsilateral to the injection site. (C-F) Photomicrographs of coronal sections at the C,-segment (C, E) and parasagittal sections at the C,-segment (D, F), of the ipsilateral spinal dorsal horn after the same injection site as in A (C, D) or B (E, F). Note the large size of cells situated in lamina V following large injections (C. D; arrows). In E, F, cells labelled in laminae IV-V are scarce.

Distribution of spino-dorsomedullary

projecting neurons

583

Table 2. Number of labelled cells at C, per spinal area in two animals injected with cholera toxin subunit B, one in the medullary dorsal reticular nucleus plus dorsal column nuclei plus nucleus of the tractus solitarius, lateral (no. 905) and another in the meduilary dorsal reticular nucleus (no. 919) Injection site ip cl ip cl

DRt + DCN + SOIL DRt

I

II

III

IV

lateral V-VI

medial V-VI

VII

x

69 29 68 38

12 3 23 11

32 II 15 6

154 44 28 14

107 35 13 7

208 8 II 8

64 46 12 14

83 87 44 42

ip, ipsilateral; cl, contralateral. DISCUSSION

The new tracer CTb was used on account of its claimed efficiency in labelling large numbers of cells retrogradely’0130,52and giving a particularly good filling of the dendritic arbors.‘0,30~3’However, since we were dealing with a new pathway, the use of two conventional tracers was thought convenient to substantiate the findings obtained with CTb. The three tracers labelled the same spinal groups in the ipsilateral cervical cord, whereas contralateral labelling at that level, and bilateral labelling caudal to C, occurred only when CTb was the marker injected. The failure of HRP tracers to produce this additional labelling does not seem to be due to insufficient transport caused by inadequate survival times. Pilot experiments showed that labelling with both HRP tracers increased from 24 to 48 h, was high and of equal intensity from 48 to 72 h, and decreased later. The use of a single survival period, 48 h, was adopted to allow numerical comparison between experiments. That an increase of retrograde labelling from 48 to 72 h of survival, has been found previously in spinothalamic cell~,~~may be ascribed to the greater distance along which the tracer was transported in that system. Again a single survival time, 72 h, was chosen for CTb since similar labelling intensities occurred between 4X h and 5 days. The large numbers of CTb labelled neurons are likely to be due to its superior sensitivity as a retrogradeiy transported marker. Previous quantitative studies of cells labelled retrogradely in the peripheral nervous system using CTb conjugated with HRP have suggested that this reagent is as equally sensitive as WGA-HRP as a transport marker. 57However, with the immunocytochemical technique of tracer detection, CTb proved to be much more sensitive than WGA-HRP, not only here but also in the spinothalamic pathway.30 A quantitative comparison with the results obtained with CTb coupled with HRP remains, however, to be done in the future. On the other hand, transynaptic transport of CTb in retrograde direction has been excluded in several systems. No transport from retrogradely labelled cell bodies to axons afferent to them has been found in sympathetic ganglia” or the hypoglossal nucleu?’ of the rat. Finally, the diffuse staining of the pial surface and the area prostrema deserves some consideration. It

has been noticed in the rat, not the cat,‘O and it has been proposed to derive from the binding of the secondary anti-mouse antibodies to endogenous rat immunoglobulins occurring in the cerebrospinal fluid.‘O Such antibodies would pass to the blood as a consequence of the penetration of the needle and would diffuse to the area prostrema through its deficient blood-brain barrier.‘O The ~eduIl~r~ dorsal reticular projection target

~u~ieu~ as a spinal

After injection sites confined to the DRt, retrogradely labelled cell bodies were found predominantly in the medial part of ipsilateral lamina I, although considerable numbers were also located in lamina X, and fewer in laminae II-IV and V-VII. When the tracer extended to the DCN and the lateral part of the nucleus of the tractus solitarius (Sol) there was an enormous increase in the number of cells in ipsilateral medial laminae IV-VI. These additional cells were probably labelled from the DCN since they had a distribution similar to that observed upon selective injections in these nucleii The moderate increase of the ipsilateral labelling in laminae IV-VI and contralateral labelling in laminae IV-VII are suggestive that these also contain a contingent of cells with medullary targets outside the DRt. The contribution of the SolL to the spinal labelling obtained after the large injections remains unclear. On the one hand, some spinal areas which project to the solitary complex25t40were not labelled since contralateral lamina I had no more labelled cells than after the small injections and the contralateral LSN remained unlabelled. On the other hand, the SolL is known to have efferent connections distinct from those of the rest of the solitary complex33 and its specific afferent inputs have not been discriminated so far. Our data suggest that the lateral part of the solitary complex is no target of lamina I and the LSN in the rat. The possibility that labeiling from the DRt depends on uptake by ascending fibres of passage is very unlikely. CTb has recently been shown to be taken up by fibres which have been lesioned by the injection. lo However, although a few spinofugal axons have been reported passing through the DRT toward the DCN,63 if they were sites of tracer uptake, tabelhng would be concentrated in iaminae IV and V, which are the confirmed afferent sources of the DCN,16 and spare lamina I. Uptake by spinothalamic or spinomesencephalic axons can be ruled out on the

.

K Fig. 4. Photomicrographs of spinal cord cells labelled from DRt (A-C, K-L) or DRt + DCN + SOIL (D-J) injections, observed in coronal sections from the cervical cord (dorsal on top, medial on the left). (A, B) Lamina 1 cells labelled with WGA-HRP (A) and CTb (B). In B roundish cells (large arrows) and mediolateral elongated cells (small arrows) are seen; large arrowheads point to ventrally directed dendrites; small arrowheads point to presumable axonic processes directed to the dorsal funiculus. (C) CTb labelled cells on the border between laminae I and II. (D) Lamina III cell labelled with WGA-HRP. Notice the dorsoventral orientation of the perikaryon and the primary dendrites. (E) Cell labelled in lamina IV with WGA-HRP. (F) Two lamina IV cells filled by CTb. (G) CTb labelled cells in the medial part of laminae V--VI. Arrows point to fusifom cells and arrowheads to roundish cells. Dashed line marks the medial contour of the dorsal horn. (H) WGA-HRP labelled fusifom cells in the same region as in G. (I, J) Cells labelled in lamina VII with WGA-HRP (I) and CTb (J), having triangular perikaryd. (K, L) CTb (K) and WGA-HRP (L) labelled cells in lamina X. 584

Distribution of spino-dorsomedullary

projecting neurons

A

Fig. 5. In A-C, camera lucida drawings of the DF lesions in experiments 946 (A), 965 (B), and 916 (C). The configuration of the LT on the operated side (left) was altered by the deformation of the spinal cord. Although the dorsal root was detached from the spinal cord, its previous location corresponded to the convexity signalled on the cord surface (*). In D and E, photomicrographs of parasagittal sections cut through the medial part of the dorsal horn of no. 965, at C,-segment on D and C,-segment on E.

585

586

D.

LIMA

Table 3. Number of cells at C,C, and C&Z, in one rat with a dorsal funicuhrs lesion at C, (no. 946) and in one control rat (no. 963) both cholera toxin subunit B injected in the medullary dorsal reticular nucleus plus dorsal column nucleus plus nucleus of the tractus solitarius, lateral C,Cz I

CC,

II-III

IV-VI

VII

x

I

II-III

IV-VI

VII

x

292 146

56 27

491 168

lo2 I32

93

0

23

181 124

135

Control

in

273 121

165 56

1807 619

245 251

385

DF lesion

:I

292 146

311 45

2136 584

495 504

456

Cl

19”

1

22

ip, ipsilateral; cl, contralateral. Ipsilateral and contralateral numbers were put together in lamina X as both halves of the lamina occurred in any 75pm parasagittal section including the central canal, so that sides could not be distinguished.

basis of the distribution

of spinal labelling.‘4~‘7~22~‘0~.“~4’ DRt spinal afferents coursing in the DF was due to

Lamina I cells labelled from the thalamu$’ or the mesencephalon3’ are predominantly contralateral and have locations along the mediolateral extent of the lamina entirely distinct from those found in this present work. Ascending tracts toward the medullary dorsal reticular nucleus

The observation of some presumable axons of lamina I neurons entering the DF suggested the possibility that spino-DRt axons could ascend there. The cuttings of the dorsal funiculus encompassed the largest part of the cuneate fasciculus in the three animals. When the cuneate fasciculus was almost entirely severed (no. 946), cells located in the superficial dorsal horn (laminae I-III) disappeared entirely from the ipsilateral side below the lesion, and were greatly diminished contralaterally. In the two other experiments in which lesions were smaller, a considerable decrease in the numbers of laminae I-III labelled cells was also noticed caudally. Since these neurons were those projecting exclusively to the DRt, it is suggested that their axons travel mostly in the cuneate fasciculus. That contralateral labelling was less affected than ipsilateral labelling in laminae I-III may be due to contralateral axons decussating rostrally to the lesion or using alternative pathways. Labelling in deeper dorsal horn cell groups (laminae IV-VI), with targets in the DRt and the DCN, was severely affected. Those ipsilateral (about 5%) and the 13% of contralateral cells which could still be labelled in laminae IV-VI after the dorsal cord lesion, were presumably neurons projecting to the DCN through the DLF, since Giesler ef aLI observed approximately 10% such cells with axons taking this route in the rat. Experiments of degeneration following dorsal cordotomy, using silver methods, have not revealed terminal changes in the DRt. In the cat using the Fink-Heimer method, Rustion?’ verified that nonprimary afferent fibres ascending in the DF terminate in the DCN. In the rat, axonal degeneration upon DF lesions was observed with the Nauta-Gygax method in the DCN, as well as in the nucleus comissuralis ipsilateralis and the medial reticular formation.13 It is probable that the previous failure to demonstrate

the techniques used. It is generally accepted that negative findings in degeneration studies are inconclusive since survival times are critical, whereas the impregnation of degenerating and terminal fibres varies with the techniques employed.’ Labelling in laminae VII and X was unaffected by DF lesions, suggesting that these cells project to the DRt through another route. Using silver impregnation, a few degenerated terminals have been observed in the DRt following lesions of the ventrolateral funiculus (VLF) in the rat6- Moreover, Rustioni and KaufmanSO verified that VLF lesions prevented labelling in laminae VII and VIII upon tracer injections in the dorsal part of the caudal medulla. Further investigations are required to elucidate the funicular trajectory of the ventral horn projection to the DRt in the rat. Functional role of the spin0 -dorsomedullary reticular pathway

Very recently Villanueva et al.” reported the involvement of the DRt in pain processing in the rat. The majority of DRt neurons were activated exclusively or preferentially by cutaneous or visceral noxious stimuli. In contrast to those of other medullary regions, DRt neurons did not respond to other sensory stimuli, such as visual, auditory or proprioceptive. GoIn addition, DRt neurons exhibited large receptive fields which often included the entire body surface. According to the present data DRt neurons receive many spinal afferents from ipsilateral lamina I. Spinothalamjc lamina I neurons, although mostly nociceptive, have characteristically small receptive fields.“,‘5 If the spinobulbar marginal cells have similarly small receptive fields, it may be conceived that inputs conveyed by several lamina I cells converge onto a single DRt neuron causing summation of their receptive fields in the target neuron. Curiously, lamina VII neurons, which contribute in a much lesser extent to the DRt input, have particularly large and complex receptive fields.‘2.‘5.35 Although some lamina VII cells are responsive to a large variety of stimuli,‘2.3s the majority are nociceptive specific.44 According to Viilanueva et ai.,@ DRt neurons respond to stimulation of contralateral receptive

Distribution of spine-dorsomedullary projecting neurons fields earlier than to that of the ipsilateral fields. Present data rather showed a small contribution of contralateral spinal neurons. However, the possibility that some primary axons activating the spino-DRt system decussate in the spinal cord cannot be ruled out. Contralateral primary afferent terminations have been referred to previously.8.27*34Namely, Light and Perl27 saw A, primary afferents terminating in the medial portion of contralateral lamina I, a location similar to that of the marginal cells that were, here, labelled retrogradely from the DRt. A better understanding of the functional significance of the spino-DRt pathway would require a more complete knowledge of the efferent connections of the DRt. The first, very recent report on this matter refers retrograde labelling of bulbar reticular neurons,26 including DRt neurons (‘personal communication of the authorsz6), following tracer injections in the mediodorsal and central median thalamic nuclei. In the light of such findings the DRt may transmit nociceptive information from the spinal cord to the medial thalamus and thus function as a relay in a polysynaptic pathway concerned with the motivational-aff~tive system of pain.38 The possible occurrence in the DF of spino-DRt fibres suggests the involvement of this funiculus in pain transmission. It may be recalled that the VLF which is the classical nociceptive ascending pathway,62 and the DLF, through which large numbers of nociceptive lamina I neurons appear to project

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may not be the only channels for pain transmission. The analgesic effects of sectioning the VLF 23v59,6’ or the DLF,3,59 are in both cases transient. It is curious that chronic pain has been relieved by electrical stimulation of the DF.4s*“*55Such effect might be attributed to the activation of low threshold primary alferents coursing in the DF, which would cause spinal segmental inhibition of pain transmission according to the gate-control theory.39 However, electrical stimulation of the DF rostra1 to a DF cut inhibits pain associated spinal reflexes below the lesion.” These data suggested that the analgesic effect mediated by the DF may be exerted through a supraspinal 10op.~’ The present results, by revealing the occurrence of a presumable nociceptive pathway connecting the spinal cord with the bulbar dorsal reticular formation through the DF, provide additional data in favour of the involvement of this funiculus in pain processing.

Acknowledgements-I am very grateful to Dr C. Sotelo, INSERM, Paris for his encouragement and technical advice, to Dr Marianne Wilkstrom, University of GBteborg, Sweden, for the kind offer of monoclonal antibodies against cholera toxin subunit B, and to Prof. A. Coimbra for the careful revision of this text. This research was supported by a grant of the Stiftung Volkswagenwerk to Prof. A. Coimbra in nartnershin with Prof. W. Ziealalnsbereer. Department of Clinicali\leuropharmacology, Eax-Planck: Institut fiir Psychiatric, Munich, and by Grant No. 87190 (IMU) from JNICT, Lisbon.

REFERENCES 1.

2. 3. 4. 5. 6. 7.

Abols I. A. and Basbaum A. I. (1981) Afferent connections of the rostra1 medulla of the cat: a neural substrate for midbrain-medullary interactions in the modulation of pain. J. camp. New&. 201, 285-297. Apkarian A. V., Stevens R. T. and Hodge C. J. (1987) The primate dorsolateral spinothalamic pathway. Sot. Neurosci. Abststr. 13, 580. Basbaum A. I., Marley N. J. E., G’Keefe J. and Clanton C. H. (1977) Reversal of morphine and stimulus-pr~u~d analgesia by subtotal spinal cord lesions. Pain 3, 43-56. Basbaum A. 1. and Fields H. L. (1984) Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. A. Rev. Neurosci. 7, 309-338. Brodal A. (1981) Neurological Anatomy in Relation to Clinical Medicine. Oxford University Press, New York. Cervero F. and Wolstencroft J. H. (1984) A positive feedback loop between spinal cord nociceptive pathways and antinociceptive areas of the cat’s brain stem. Pain 20, 125-138. Chaouch A., Menetrey D., Binder D. and Besson J. M. (1983) Neurons at the origin of the medial component of the bulbopontine spinoreticular tract in the rat: an anatomical study using horseradish peroxidase retrograde transport.

J. camp. Neuroi. 214, 309-320. 8. Culberson J. L., Haines D. E., Kimmel D. L. and Brown P. B. (1979) Contralateral projection of primary afferent fibres to mammalian spinal cord. Expl Neural. 64, 83-97. 9. De Olmos J., Hardy H. and Heimer L. (1978) The afferent connections of the main and the acessory olfactory bulb formations in the rat: an experimental HRP-study. J. camp. Neural. 181, 213-244.

10. Ericson H. and Blomqvist A. (1988) Tracing of neuronal connections with cholera toxin subunit B: light and electron microscopic immunoh~stochemist~ using monoclonal antibodies. J. Neurosci. Me&. 24, 225235. Il. Ferrington D. G., Sorkin L. S. and Willis W. D. Jr (1987) Responses of spinothalamic tract cells in the superficial dorsal horn of the primate lumbar spinal cord. J. Pbysioi. 388, 681-703. 12. Fields H. L., Clanton C. H. and Anderson D. (1977) Somatosensory properties of spinoreticular neurons in the cat. Brain Res. 120, 49-66. 13. Ganchrow D. and Bernstein J. J. (1981) Projections of caudal fasciculus gracilis to nucleus gracilis and other medullary structures, and Clarke’s nucleus in the rat. Brain Res. 205, 383-390. 14. Giesler G. J. Jr, Menetrey D. and Basbaum A. I. (1979) Differential origins of spinothalamic tract projections to medial and lateral thalamus in the rat. J. camp. Neurol. 184, 107-126.

15. Giesler G. J. Jr, Yezierski R. P., Gerhart K. D. and Willis W. D. (1981) Spinothalamic tract neurons that project to medial and/or lateral thalamic nuclei: evidence for a physiologically novel population of spinal cord neurons. J. Neurophysiol. 46, 1285-l 308. 16. Giesler G. J. Jr, Nahin R. L. and Madsen A. M. (1984) Postsynaptic dorsal column pathway of the rat. I. Anatomical studies. J. Neurophysioi. 51, 260-275.

588

D. LIMA

17 Granum S. L. (1986) The spinothalami~ system of the rat. 1. Locations of cells or origin. J. crimp. Neurol. 247, 1599180. 18 Hylden J. L. K., Hayashi H. and Bennett G. J. (1986) Lamina I spinomesencephalic neurons in the cat ascent via the dorsolateral funiculi. Somatosensory Res. 4, 3141, 19 Itoh K., Konishi A., Nomura S., Mizuno N., Nakamura Y. and Sugimoto T. (1979) Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method. Brain Res. 175, 341-346. 20. Jones M. W., Hodge C. J. Jr, Apkarian A. V. and Stevens R. T. (1985) A dorsolateral spinothalamic pathway in cat. Brain Res. 355, 188193. fixative of high osmolality for use in electron microscopy. J. 21. Karnovsky M. J. (1965) A formaldehyde-glutaraldehyde Cell Bin!. 27, 137/A. 22 Kemplay S. K. and Webster K. E. (1986) A qualitative and quantitative analysis of the distributions of cells in the spinal cord and spinomedullary junction projecting to the thalamus of the rat. Neuroscience 17, 769-789. 23 Kennard M. A. (1954)The course of ascending fibres in the spinal cord of the cat essential to the recognition of painful stimuli. J. camp. Neurol. 100, 511-524. 24. Kevetter G. A., Haber L. H., Yezierski R. P., Chung J. M., Martin R. F. and Willis W. D. (1982) Cells of origin of the spinoreticular tract in the monkey. J. camp. Neural. 207, 61.-74. 25. Leah J., Menetrey D. and De Pommery J. (I 988) Neuropeptides in long ascending spinal tract cells in the rat: evidence for parallel processing of ascending information. Neuroscience 24, 195-207. 26. Lechner J., Carstens E., Leah J. and Zimmermann M. (1988) Brainstem projections to medial thalamus and hypothalamus. Abstracts from the I Ith Annual Meeting of the European Neuroscience Association. 74.24, 284. 27. Light A. R. and Per1 E. R. (1979) Reexamination of the dorsal root projection to the spinal dorsal horn including observations on the differential termination of coarse and fine tibres. J. camp. Neurul. 186, 117-132. 28. Lima D. and Coimbra A. (1983) The neuronal population of the marginal zone (lamina 1) of the rat spinal cord. A study based on reconstructions of serially sectioned cells. Anut. Embryol. 167, 2733288. 29. Lima D. and Coimbra A. (1985) Marginal neurons of the rat spinal cord at the origin of a spinobulboreticular projection. Newosci. Left. 22, S9. 30. Lima D. and Coimbra A. (1988) The spinothalamic system of the rat: structural types of retrogradely labelled neurons in the marginal zone (lamina I). Neuroscience 27, 215-230. 31. Lima D. and Coimbra A. (1989) Mo~hological types of spinomesenc~phali~ neurons in the marginal zone (Iamina I) of the rat spinal cord, as shown after retrograde lahelling with cholera toxin subunit B. J. camp. Neurol. 279,327-339. 32. Lima D. and Coimbra A. (1990) Structural types of marginal (lamina I) neurons projecting to the dorsal reticular nucleus of the medulla oblongata. Neu~~~cjence 34, 591606. 33. Loewy A. D. and Burton H. (1978) Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat. J. romp. Neuroi. 181, 421.-450. 34. Matsushita M. and Tanami T. (1983) Contralateral termination of primary a&rent axons in the sacral and caudal segments of the cat, as studied by anterograde transport of horseradish peroxidase. J. camp. Neural. 220, 206-218. 35. Maunz R. A.. Pitts N. G. and Peterson B. W. (1978) Cat spinoreticular neurons: locations, responses and changes in responses during repetitive stimulation. Brcrin Res. 148, 365-379. 36. McMahon S. B. and Wall P. D. (1983) A system of rat spinal cord lamina I cells projecting through the contralateral dorsolateral funiculus. J. camp. Neural. 214, 217-223. 37. McMahon S. B. and Wall P. D. (1985) Electrophysiological mapping of brainstem projections of spinal cord lamiaa I cells in the rat. Brain Res. 333, 19-26. 38. Melzack R. and Casey K. L. (1968) Sensory, motivational and central control determinations of pain. In The Skin Senses (ed. Kenshalo D. R.), pp. 4233443. Thomas, Springfield. 39. Melzack R. and Wall P. D. (1965) Pain mechanisms: a new theory. Science 150, 971-979. 40. Menetrey D. and Basbaum A. I. (1987) Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and visceroviscerai reflex activation. .J. camp. Neural. 255, 439450. 41. Menetrey D., Chaouch A., Binder D. and Besson J. M. (1982) The origin of the spinomesencephalic tract in the rat: an anatomical study using the retrograde transport of horseradish peroxidase. J. camp. Ncurol. 206, 193-207. 42. Menttrey D., Roudier F. and Besson J. M. (1983) Spinal neurons reaching the lateral reticular nucleus as studied in the rat by retrograde transport of horseradish peroxidase. J. camp. Neurol. 220, 439452. 43. Mesulam M. M. (1982) Principles of horseradish peroxidase neurohistochemistry and their applications for tracing neural pathways-axonal transport, enzyme histochemistry and tight microscopic analysis. In Tracing Neural Connecfions with Horseradish Peroxiduse (ed. Mesulam M. M.), pp. I-151. John Wiley & Sons, New York. 44. Molinari H. H. (1982) The cutaneous sensitivity of units in laminae VII and VIII of the cat. Brain Res. 234, 1655169. 45. Nashold B. S. Jr and Friedman, H. (1972) Dorsal column stimulation for control of pain. Preliminary report on 30 patients. J. Neurosurg. 36, 590-597. 46. Paxinos G. and Watson C. (1982) The Rat Brain in Sfereofaxic Coordinates. Academic Press, New York. 47. Peschanski M. and Besson J. M. (1984) A spino-reticuio-thalamic pathway in the rat: an anatomical study with reference to pain transmission. Neuroscience 12, 165-178. 48. Rexed B. (1952) The cytoarchitectonic organization of the spinal cord in the cat. J. camp. Neural. 96, 415-495. 49. Rustioni A. (1974) Non-primary afferents to the cuneate nucleus in the brachial dorsal funiculus of the cat. Brain Rex. 75, 247-259.

50. Rustioni A. and Kaufman A. B. (1977) Identification of cells of origin of non-primary afferents to the dorsal column nuclei of the cat. ExpZ Brain Res. 27, l-14. 51. Saade N., Atweh S. F., Tabet M. S. and Jabbur S. J. (1985) Inhibition of nociceptive withdrawal flexion reflexes through a dorsal column-brainstem-spinal loop. Bruin Res. 335, 306308. 52. Sawchenko P. E. and Gerfen C. R. (1985) Plant lectins and bacterial toxins as tools for tracing neuronal connections. Trends Neurosci. 8, 378-384. 53. Schwab M. E.. Suda K. and Thoenen H. (1979) Selective retrograde transsvnaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport.‘J. Cell Biol. 82, 798-810. _ _ 54. Shealy C. N., Mortimer J. T. and Reswick J. B. (1967) Electrical inhibition of pain by stimulation of the dorsal column: preliminary clinical report. Anestk. Ana(. 46, 489-491.

Distribution of spino-dorsomedullary

projecting neurons

589

55. Shealy C. N., Mortimer J. T. and Hagfors N. R. (1970) Dorsal column electroanalgesia. J. Neurosurg. 32, 560-564. 56. Shokunbi M. T., Hrycyshyn A. W. and Flumerfelt B. A. (1985) Spinal projections to the lateral reticular nucleus in the rat: a retrograde labelling study using horseradish peroxidase. J. camp. Neurol. 239, 216226. 57. Trojanowski J. Q., Gonatas J. 0. and Gonatas N. K. (1982) Horseradish peroxidase (HRP) conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP. Brain Res. 231, 33-50. 58. Ugolini G.. Kuvners H. G.. Simmons A. and O’Hanlon S. P. (19861 Use of hernes simnlex virus as retrograde tr&sneuronal tracer: labelling of interneurones to hypoglossal mdtoneurones. Neur&i. Z_& Suppl. 26, S239. 59. Vierck C. J. and Luck M. M. (1979) Loss and recovery of reactivity to noxious stimuli in monkeys with primary spinothalamic cordotomies, followed by secondary and tertiary lesions of other cord sectors. Bruin 102, 233-248. 60. Villanueva L., Bouhassira D., Bing Z. and Le Bars D. (1988) Convergence of heterotopic nociceptive information onto subnucleus reticularis dorsalis (SRD) neurons in the rat medulla. J. Neurophysiol. 60, 98&1009. 61. White J. C. and Sweet W. H. (1969) Pain and the Neurosurgeon. Thomas, Springfield. 62. Willis W. D. (1985) The pain system. The neural basis of nociceptive transmission in the mammalian system. In Pain and Headache (ed. Gilder&erg Ph. L.), Vol. 8. Karger, New York. 63. Zemlan F. P., Christiana M. L., Kow L.-M. and Pfaff D. W. (1978) Ascending tracts of the lateral columns of the rat spinal cord: a study using the silver impregnation and horseradish peroxidase techniques. Expl Neural. 62, 298-334. (Accepted 9 August 1989)