The origin of brainstem noradrenergic and serotonergic projections to the spinal cord dorsal horn in the rat.

Somatosensory & Motor Research

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The Origin of Brainstem Noradrenergic and Serotonergic Projections to the Spinal Cord Dorsal Horn in the Rat Geoffrey C. Kwiat & Allan I. Basbaum To cite this article: Geoffrey C. Kwiat & Allan I. Basbaum (1992) The Origin of Brainstem Noradrenergic and Serotonergic Projections to the Spinal Cord Dorsal Horn in the Rat, Somatosensory & Motor Research, 9:2, 157-173, DOI: 10.3109/08990229209144768 To link to this article: http://dx.doi.org/10.3109/08990229209144768

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The Origin of Brainstem Noradrenergic and Serotonergic Projections to the Spinal Cord Dorsal Horn in the Rat Geoffrey C. Kwiat' and M a n I. Basbaum2

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Keck Center for Integrative Neuroscience and Departments of Anatomy and Physiology, University of California at San Francisco, San Francisco, California 94143 Abstract Although it has been proposed that the locus coeruleus is the predominant, if not exclusive, brainstem origin of the noradrenergic innervation of the spinal dorsal horn, pharmacological studies argue otherwise. In this study we made localized injections of the retrograde tracer wheatgerm agglutinin conjugated to apo-horseradish peroxidase gold (WGA:apoHRP-Au), in conjunction with immunocytochemical labeling for tyrosine hydroxylase (TH) or serotonin (5-HT), to identify the brainstem source of the noradrenaline (NA) and 5-HT innervation of the dorsal horn of the rat. Our studies were concentrated in the C5 spinal segment. The pattern of labeling was only studied in animals in which the tracer injection was restricted to the dorsal horn. In these rats, TH-immunoreactive neurons in widespread regions of the brainstem, including the locus coeruleus, subcoeruleus, AS, and A7 cell groups, were found to project to the dorsal horn. In terms of absolute numbers of double-labeled cells, no one noradrenergic cell group predominated. As expected, dorsal-horn-projecting 5-HT-immunoreactive neurons were found within the 5-HTpopulations of the rostroventromedial medulla and caudal pons, including the nucelus raphe magnus, nucleus paragigantocellularis (PGi), and ventral portions of the nucleus gigantocellularis (Gi). The majority of retrogradely labeled 5-HT-immunoreactive cells were, however, located off the midline, in the ipsilateral PGi and ventral Gi. Finally, a large number of retrogradely labeled, non-5-HT cells were found intermingled among the 5-HT cells of this region. Our results provide evidence that the noradrenergic regulation of nociceptive transmission at the spinal cord level arises from direct spinal projections of several brainstem noradrenergic cell groups.

Key words antinociception, noradrenaline (NA), serotonin (5-HT), dorsal horn, locus coeruleus, subcoeruleus, A5, A7, immunocytochemistry

The analgesia produced by opiate injection into or electrical stimulation of the midbrain periaqueductal gray (PAG) is hypothesized to result from the activation of brainstem serotonin (5-HT) and noradrenaline (NA) neurons that project to and inhibit the firing of spinal cord nociresponsive neurons (for reviews, see Basbaum et al., 1983; Basbaum and Fields, 1984; Proudfit, 1988; Reichling et al., 1988). This hypothesis is supported by several observations: Intrathecal administration of 1. Present address: Department of Anatomy and Developmental Biology, University College London, Gower Street, London WClE 6BT, United Kingdom. 2. To whom all correspondence should be addressed, at Department of hatomy, BOX 0452, University of California at sari Francisco, San Francisco, California 94143.

5-HT antagonists reverses the antinociception evoked by electrical stimulation of or opiate injection into the PAG (Yaksh, 1979; Aimone et al., 1987); intrathecally administered 5-HT (Yaksh and Wilson, 1979) or a2adrenergic agonists (Kuraishi et al., 1979; Reddy et al., 1980; Howe et al., 1983) produce a dose-dependent analgesia; and local iontophoretic injection of 5-HT or NA inhibits dorsal horn neuronal responses to noxious stimuli (Headley et al., 1978; Satoh et al., 1979; Howe and Zieglgansberger, 1987). Spinal noradrenergic-mediated antinociceptive effects can be produced not only by stimulation of the pAG, but by Or glutamate in other brainstem regions, including the rostroventromedial medulla (RVM) (Aimone et al., 1987), lateral

Somatosensory and Motor Research, Vol. 9. No. 2, 1992, pp. 157-173

Accepted January 29, 1992

157


KWIAT AND BASBAUM

reticular nucleus (Gebhart and Ossipov, 1986), and several pontine catecholamine cell groups (Jones and Gebhart, 1986; Miller and Proudfit, 1990; Burnett and Gebhart, 1991). Although these studies implicate all of the spinally projecting brainstem noradrenergic cell groups (locus coeruleus [LC], subcoeruleus [SC], A5, and A7) in the generation of a-adrenergic-mediated antinociceptive effects, it is not known whether these effects involve direct activation of bulbospinal noradrenergic neurons in the immediate area, or indirect activation via connections with other brainstem noradrenergic cell populations that project to and inhibit dorsal horn nociresponsive neurons. That noradrenergic neurons of the LC, SC, A5, and A7 cell groups project to the spinal cord has been clearly demonstrated (Westlund and Coulter, 1980; Westlund et al., 1983; Loewy et al., 1986; Kwiat and Basbaum, 1989; Clark and Proudfit, 1991~).It is not established, however, whether each of these brainstem noradrenergic cell groups specifically projects to the dorsal horn. From the results of retrograde tracing combined with selective lesions with the neurotoxin DSP-4, Lyons et al. (1989) concluded that the LC is the predominant, if not exclusive, source of the dorsal horn NA innervation, and that the A5 and A7 cell populations innervate the ventral and intermediolateral (IML) gray matter. Consistent with that conclusion, these authors demonstrated a direct noradrenergic coeruleospinal projection to the dorsal horn using the Phuseolus vulgaris leucoagglutinin (PHA-L) anterograde tracing technique (Fritschy et al., 1987; Fritschy and Grzanna, 1990). The DSP-4 results, however, do not rule out a contribution of the SC or of the A5 and A7 cell groups to the NA innervation of the dorsal horn. In fact, although there is general agreement that the A5 cell group is the origin of the NA innervation of the IML (Loewy et al., 1979; Byrum and Guyenet, 1987), the finding that the LC projects predominantly to the dorsal horn is at odds with earlier studies, which concluded that the LC projects either predominantly to the ventral horn (Nygren and Olson, 1977) or diffusely to both dorsal and ventral horn (Westlund and Coulter, 1980; Holstege and Kuypers, 1982). Another approach to determining the relative contribution of different brainstem cell populations to the dorsal horn innervation is to make restricted injections of a suitable retrograde tracer into the dorsal horn. To this end, Clark et al. (1991) used the retrograde transport of fluorogold from spinal cord and demonstrated that the A7 cell group is, in fact, a source of noradrenergic axons to the dorsal horn. They also found that the extent to which different brainstem cell groups contribute noradrenergic axons to the dorsal horn is substraindependent. Since fluorogold is taken up and transported 158

by fibers of passage (Cliffer and Giesler, 1988) it could be argued that the retrogradely labeled cells recorded in these studies were en route to the IML, particularly since the injection sites included the entire dorsal horn. To address that possibility, we have re-examined this question using the retrograde tracer wheatgerm agglutinin conjugated to apo-horseradish peroxidase gold (WGA:apoHRP-Au) (Basbaum and MenCtrey, 1987). This tracer has characteristics that make it ideal for making small restricted injections. It is highly viscous, so that diffusion is minimal, and it is sufticiently sensitive that even small injections result in large numbers of retrogradely labeled cells. The particulate silver reaction product used to visualize the tracer in cell bodies is particularly convenient for double-labeling studies. Finally, there appears to be very limited uptake of the tracer by axons of passage or damaged axons. In the present study, we made restricted WGA:apoHRP-Au injections into a single segment of cervical dorsal horn to study the dorsal horn projections of brainstem tyrosine hydroxylase (TH)-immunoreactive and 5-HT-immunoreactive cells. As expected, the distribution of 5-HT cells projecting to the dorsal horn was localized to the nucleus raphe magnus (NRM) and adjacent reticular formation. In contrast, even with very small dorsal horn injections at a single spinal level, we found that retrogradely labeled TH-immunoreactive cells were widely distributed throughout the brainstem NA cell groups. A preliminary report of this work has been published (Kwiat and Basbaum, 1990). MATERIALS AND METHODS T o facilitate making restricted injections of WGA:apoHRP-Au, we chose to study the C5 segment, which has a large dorsal horn and, as part of the cervical enlargement, should receive a large projection from brainstem sites. Under pentobarbital anesthesia (60mgl kg), laminectomies were performed on 250 to 300-g male Sprague-Dawley rats (Bantin-Kingman) to expose the C5 spinal cord segment. The dura was slit, and 0.1-0.3 p1 of a solution of WGA:apoHRP-Au was injected with a glass micropipette (tip diameter 30-50 pm). The micropipette was secured to a stereotaxic manipulator and connected, via polyethylene tubing, to a 10-p1 Hamilton syringe. The micropipette and syringe were filled with mineral oil, and then the WGA:apoHRP-Au was drawn into the pipette tip. A single penetration of the spinal cord was made just medial to the dorsal root entry zone. To minimize diffusion along the pipette track, the tracer was injected over a period of several minutes. The overlying muscle and skin were sutured, after which the rats were returned to their cages and allowed to recover.


NA AND 5-HT PROJECTIONS TO DORSAL HORN

After a survival time of 5-7 days to allow for retrograde transport of the WGA:apoHRP-Au, the rats were deeply anesthetized and transcardially perfused with 150 ml of a 37°C solution of 0.05 M phosphatebuffered saline, followed by 500 ml of a 4°C fixative solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains and cervical spinal cords were quickly removed, postfixed in the same fixative for 2-3 hr at 4"C, and then placed in a phosphatebuffered 30% sucrose solution for the purpose of cryoprotection. Frozen sections 30 and 50 Fm thick were cut through the brainstem and spinal cord injection site, respectively. Sections were silver-enhanced to visualize the retrogradely transported WGA:apoHRPAu, according to a previously published procedure (Basbaum and MenCtrey, 1987). Adjacent silver-enhanced sections through the brainstem were then processed for either TH (Eugene Tech, Allendale, NJ) or 5-HT (Incstar, Stillwater, MN) immunoreactivity according to the avidin-biotin complex method (Hsu et al., 1981), with diaminobenzidine (DAB) as chromogen. The distributions of retrogradely labeled and immunoreactive cells were plotted on camera lucida drawings of the brainstem sections at intervals of approximately 200 pm (Fig. 3, below). For TH-immunostained sections, the drawings extended from the midmedullary region to the caudal midbrain. More caudal sections were not included because they did not contain neurons retrogradely labeled from the dorsal horn. For 5-HT-immunoreacted sections, plots included only the region of the NRM and adjacent reticular formation, from the caudal extent of the seventh nerve nucleus to the caudal pole of the superior olive (Fig. 7, below). This region corresponds to the area enclosed within the dorsal, lateral, and ventral boundaries of the 5-HT distribution in the RVM and caudal pons, and was chosen because it is the region from which analgesia is best elicited by electrical stimulation (Zorman et al., 1982). This region was further divided into a midline zone, and into ipsilateral and contralateral regions (relative to the injected site), as depicted below in Figure 7B. The lateral regions extended to the lateral edge of the pyramids, or, where the pyramids were absent rostrally, to an equivalent distance from the midline. Although a large number of rats received spinal cord injections, it proved to be very difficult to completely prevent spread of the tracer beyond the dorsal horn. Our analysis is therefore limited to results from four rats in which the WGA:apoHRP-Au injection was entirely restricted to the dorsal horn. In all four cases, retrograde labeling was combined with TH-immunocytochemical labeling. In two of the four cases, in adjacent sections, retrograde labeling was combined with 5-HT immunocytochemistry. Because of the small

numbers of animals, it was not possible to perform statistical analyses. RESULTS Znjection Sites

Composite drawings of the maximal extent of the WGA:apoHRP-Au dorsal horn injections are shown in Figure 1. In all cases the tracer was concentrated in the superfical laminae of the dorsal horn. Although the injection sites usually included a thin extension of tracer into the neck of the dorsal horn, possibly caused by respiratory movement during the injection, in no case did the injection extend ventrally, beyond the neck of the dorsal horn. The injection in animal 150 was somewhat more medial, and the injection in 145 more lateral, to the injection of animal 138, which was the most centrally located in the dorsal gray matter. Some tracer was invariably deposited within the white matter overlying the dorsal horn, along the path of the micropipette. The photomicrograph of the silver-enhanced injection site of case 138 reveals the characteristic dense core of the tracer surrounded by particulate labeling (Fig. 1B); the latter consists largely of transported tracer. Retrogradely labeled cells, both in the immediate vicinity of the injection and at varying distances from the injection, are also visible. Some of these, of course, may have been labeled because the tracer was taken up by damaged dendrites. General Pattern of Retrograde Labeling

Although the placement and volume of WGA:apoHRPAu injected differed slightly among the four animals, the patterns of retrograde labeling in the brainstem were similar. The distribution of labeled cells for the case with the largest number of neurons (138) is presented (Fig. 3). In all cases, the regions containing the highest concentration of retrograde cell labeling were the RVM and caudal ventromedial pons (Figs. 2, 3e3k), including the NRM medially and the gigantocellular reticular nucleus pars alpha (Gia) and paragigantocellular reticular nucleus (PGi) laterally. Few retrogradely labeled cells were found in the raphe pallidus and obscurus nuclei. Retrogradely labeled cells were also noticeably absent from regions of the reticular formation dorsolateral to the NRM (Figs. 2,3g-3i). Many retrogradely labeled cells were also found in or near the A5, A7, LC, and SC NA cell groups. The ventrolateral medulla, in the vicinity of the A1 and C1 catecholamine cell groups (Figs. 3a-3f) and the dorsally situated C2 cell group (Figs. 3e, 3f), contained a small number of projecting cells. Finally, relatively small to moderate num159

KWIAT AND BASBAUM


143

145

150

(A)

FIGURE 1. (A) Camera lucida drawings of the silver-enhanced WGA:apoHRP-Au cervical dorsal horn injection sites for four dserent animals. The blackened areas represent the total extent of the deposited tracer. The drawings shown are composite drawings of individual 50-pm sections through the rostrocaudal extent of the injections. Since variations in tissue shrinkage of the individual sections made it difficult to overlay adjacent sections precisely, the sizes of the tracer injections shown are likely to be overestimates. (B) Photomicrograph of a single silver-enhanced 50-pm section near the middle of the rostrocaudal extent of the WGA:apoHRP-Au dorsal horn injection in animal 138. The injection is most densely concentrated in the superficial laminae of the dorsal horn. Retrogradely labeled cells can be seen in the ventral part of the dorsal horn and in the intermediate regions of the gray matter, at varying distances from the injection site. DC, dorsal columns; DLF, dorsolateral funiculus. Calibration bar: 100 pm.

bers of retrogradely labeled cells were found bilaterally in the dorsal medulla, in the region of the vestibular nuclei, in three of four animals (138, 145, 150; Figs. 3b-3f). Distribution of TH-Zmmunoreactive Projecting Cells

The pattern of labeling of brainstem TH-immunoreactive cells shown in Figure 3 is essentially identical to previously published reports of TH- and dopamine-p-hydroxylase (DBH)-immunoreactive cells (Westlund et al., 1983;Kalia el al., 1985). Briefly, TH-immunoreactive cells were widely distributed in the brainstem: in the ventrolateral medulla (A1,CI) and dorsomedial medulla (A2,C2); in the ventrolateral pons (As)and dorsolateral pons (LC, SC); and in a region ventral to the ventrolateral 160

tip of the superior cerebellar peduncle at the level of the pons-midbrain junction (A7). At rostra1 levels of the A5 cell group, the ventral SC and dorsal A5 cells formed a dorsoventral string of labeled cells (Figures 31, 3m). The dorsal horn injection of WGA:apoHRP-Au in animal 138 (Figs. lA, 1B) resulted in double labeling of a widely distributed population of brainstem THimmunoreactive cells that projected to the dorsal horn (Fig. 3, Table 1). In fact, retrogradely labeled THimmunoreactive cells were found in all brainstem NA cell groups projecting to the spinal cord-that is, in the LC, SC, A5, and A7 cell groups (Figs. 3,4-6). The A5 cell group in animal 138 contained the largest total number of retrogradely labeled TH-immunoreactive cells, followed closely by the LC and SC, which con-

FIGURE2. This darkfield photo montage of the rostroventral medulla at the level of the seventh nerve nucleus (VII)demonstrates the fairly restricted distribution pattern of retrogradely labeled cells resulting from the WGA:apoHRP-Au dorsal horn injection in animal 138. The retrogradely labeled cells appear white in this darkfield image. Retrogradely labeled cells are located in the nucleus raphe magnus (Mag), nucleus paragigantocellularis lateralis (PGL), and the ventral region of the nucleus gigantocellularis (Gi). Note the almost complete absence of retrogradely labeled cells dorsal to these areas. The vertical slit in the pyramid on the left side marks the side contralateral to the dorsal horn injection of the WGA:apoHRP-Au. Calibration bar: 100 pm.



KWIAT AND BASBAUM

FIGURE3. These schematic camera lucida drawings of brainstem sections show the distribution of TH-immunoreactive retrogradely labeled cells (triangles) and non-TH-immunoreactive retrogradely labeled cells (squares) resulting from the WGA:apoHRP-Au dorsal horn injection in animal 138. TH-immunoreactive cells that were not retrogradely labeled are also indicated for comparison (circles). Ipsilateral to the injection site is on the left of each drawing, which depicts the location of labeled cells in a single 30-pm section. Small symbols represent a single cell; large symbols represent five cells. The single stars in k and 1 represent SO TH-immunoreactive cells in the LC. A l , A1 noradrenergic cell group; A2, A2 noradrenergic cell group; C1, C1 adrenergic cell group; C2, C2 adrenergic cell group; 10, inferior olivary nucleus; Sol, nucleus tractus solitarii; Pa, raphe pallidus nucleus; 12, hypoglossal nucleus; Sp5, spinal trigeminal nucleus; sp5, spinal tract of the trigeminal nucleus; Mag, nucleus raphe magnus; VII, seventh nerve nucleus; VIIn, seventh cranial nerve; AS, AS noradrenergic cell group; SO, superior olivary nucleus; LC, locus coeruleus; SC, subcoeruleus; SCP, superior cerebellar peduncle; A7, A7 noradrenergic cell group.

162


NA AND 5-HTPROJECTIONS TO DORSAL HORN

A

TH project non-TH project TH

FIGURE 3. (Continued)

tained approximately equal numbers of double-labeled cells. The A7 cell group contained a few double-labeled cells (Table I). TH-immunoreactive projection cells were commonly found in the NA cell population that extends from the ventrolaterally situated A5 cell group to the dorsally situated LC and SC (Fig. 3m). Unless a clear association with the more ventral A5 cells (Figs. 3h-31) was evident, these cells were considered to be part of the ventral extent of the SC (see Paxinos and Watson, 1986). Only a rare retrogradely labeled THimmunoreactive cell was found in the medullary nor-

adrenergic (A1,A2)and adrenergic (Cl ,C2)cell groups (Fig. 3). Approximately one-third of the total TH-immunoreactive cells in the SC (31%, combined ipsilateral and contralateral) and 16% of the total number of THimmunoreactive cells in the A5 cell group (combined ipsilateral and contralateral) were retrogradely labeled by an injection of tracer into the dorsal horn of Cs. Although the LC contained a comparable number of retrogradely labeled TH-immunoreactive cells to that of the SC,a considerably smaller proportion of its total 163

KWIAT AND BASBAUM

TABLE1. Distribution of TH-Immunoreactive Cells Retrogradely Labeled from the Cs Dorsal Horn 138

TH A5

LC

sc


A7

Ipsilateral Contralateral Ipsi1ateral Contralateral Ipsilateral Contralateral Ipsilateral Contralateral

TH

retrograde

%

TH

104 123 262 248 37 48 52

24 12 17 12 15 11 2 6

23

59 73 177 214 33 17 19 26

55

10

6 5

40 23 4 11

145 TH retrograde 9 7 25 14 10 5

7 8

%

TH

15 10 14 6 30 29 37 3

90 79 236 235 37 24 31 34

150 TH retrograde

% 11 13 2 3 24 12 10 6

Note. The numbers 138, 145, and 150 refer to three of the spinal cord WGA:apoHRP-Au tracer injections schematically depicted in Figure 1. Numbers are actual counts of cells made from sequential series of brainstem sections, separated by approximately 200 km. The dserences among animals in the number of TH-immunoreactive cells counted in the respective cell groups may be a consequence, in part, of the method for selecting brainstem series; more or less of a given cell group could be included in a given series. The results of animal 143 are excluded from the table because the total number of retrogradely labeled cells in this animal was very small, compared to those of the other three animals.

TH-immunoreactive population projected (Table 1). Finally, non-TH-immunoreactive retrogradely labeled cells were intermingled among all of the TH-immunoreactive cell groups (Figs. 3-5). Although there were differences in absolute numbers, the relative distribution of TH projection neurons was similar in all rats studied (Table 1). In all animals (including case 143, in which only a small number of cells were retrogradely labeled), we observed TH-immunoreactive projection neurons in the LC, SC, AS, and A7 cell groups. One exception to the general pattern of labeling was that in animal 145, in which the dorsal horn injection was more laterally located than in the other animals, the A7 cell group contained a proportionately greater number of double-labeled cells (Table 1).

Distribution of 5-HT-Immunoreactive Projecting Cells The distribution of retrogradely labeled 5-HT- and non5-HT-immunoreactive cells in the RVM and caudal ventromedial pons was plotted for animals 138 and 150 (Table 2). The results were comparable for both rats. Figure 7A illustrates the projection pattern for rat 138. Although retrogradely labeled cells were bilaterally distributed, the ipsilateral ventral gigantocellular reticular (Gi) and lateral paragigantocellular reticular (PGL) nuclei contained approximately twice as many projection cells (both 5-HT and non-5-HT) as did the contralateral side. The number of projection cells near the midline region was comparable to the number contralateral to the injection (Table 2). Figure 8 shows examples of 164

retrogradely labeled 5-HT- and non-5-HT-immunoreactive neurons in the ipsilateral PGL and NRM. Overall, only 9% of the total number of 5-HTimmunoreactive cells counted in animal 138 were also retrogradely labeled (Table 2). The majority of the retrogradely labeled 5-HT-immunoreactive cells were found in the ipsilateral ventral Gi and PGL. In this region, about 18% of the 5-HT-immunoreactive cells were retrogradely labeled. Only 4% of the 5-HT-immunoreactive cells counted in both the midline and contralateral regions were retrogradely labeled from the dorsal horn. Large numbers of the retrogradely labeled cells in midline and lateral regions were not 5-HT-immunoreactive. In fact, 84% of all the retrogradely labeled cells counted in animal 138 were not immunoreactive for 5-HT. With respect to the number of retrogradely labeled cells in midline and lateral regions, 74% of the retrogradely labeled cells in the ipsilateral region and 94% and 89% of the retrogradely labeled cells in the midline and contralateral regions, respectively, were not 5-HT-immunoreactive. . Far fewer 5-HT-immunoreactive cells in animal 150 were retrogradely labeled; this may have resulted from the fact that this animal had far fewer total retrogradely labeled cells than did animal 138. The pattern of 5-HT- and non-5-HT-immunoreactive projecting cells in the two rats was, however, very similar. In both animals, over 70% of all 5-HT-immunoreactive projecting cells counted were located ipsilaterally ;the relative numbers of 5-HT- and non-5-HT-immunoreactive projecting cells in the midline and contralateral regions were also comparable (Table 2).



FIGURE4. Double-labeled TH-immunoreactive retrogradely labeled cells in the A5 cell group. (A) Low-power darkfield photomicrograph shows the proximity of retrogradely labeled cells to the superior olive (SO). The midline is to the left in this photomicrograph. Open arrows point to retrogradely labeled cells that were not TH-immunoreactive.(B and C) Higherpower brightfield photomicrographsof the cells indicated by the solid arrow and by the arrowhead in A, respectively. This magnification reveals that these cells are immunoreactive for TH (i.e., contain diffuse DAB reaction product) and also contain silver particles indicative of retrogradely transported tracer (black dots). TH-immunoreactive cells that do not contain silver particles are also indicated in B (arrows). Calibration bar in A: 100 pm. Calibration bar in B (for both B and C): 10 pm. 165


FIGURE5. TH-immunoreactive and non-TH-immunoreactive retrogradely labeled cells in the locus coeruleus. A and B are darklield and brightfield images, respectively, of the same field. Silver particles indicating the presence of retrograde tracer appear white in A. In B, the TH immunoreaction product is diffusely located throughout the labeled somata and proximal dendrites; although it is not easily seen at this magnification, the silver retrograde label appears as black punctate particles. A higher magnification of the lower half of the field in A and B is shown in C. Arrowheads in A, B, and C point to retrogradely labeled but not TH-immunoreactive cells. Arrows in these images point to double-labeled cells. Calibration bars in B (for both A and B) and C: 50 pm.



FIGURE 6. Double-labeled cells in the subcoeruleus. A and B are darkfield and brightfield photomicrographs, respectively, of the same field. The three clearly visible retrogradely labeled cells in A (arrows) are all also TH-immunoreactive (B, arrows). TH-immunoreactive cells that were not retrogradely labeled are also present (arrowhead). Calibration bar in B (for both A and B): 50 bm.

TABLE2. Distribution of 5-HT and Non-5-HT Cells in the RVM and Caudal Ventromedial Pons Retrogradely Labeled from the C5 Dorsal Horn 138

Ipsilateral Midline Contralateral Combined

150

5-HT retrograde

Non-5-HT retrograde

(%I

(%I

Total 5-HT

5-HT retrograde (9%)

Non-5-HT retrograde

Total 5-HT 495 332 507

89 (18) 15 (4) 19 (4)

249 (74) 247 (94) 149 (89)

272 208 234

12 (4) 3 (1) 2 (1)

27 (70) 49 (94) 14 (88)

645 (84)

714

17 (2)

90 (84)

1334

123 (9)

(%I

Nore. The numbers 138 and 150 refer to spinal cord dorsal horn injections of WGA:apoHRF’-Au (Fig. 1) in two animals. Numbers are actual counts of cells made from sequential series of brainstem sections, separated by approximately 200 pm. The numbers in parentheses beneath the columns headed “5-HT retrograde” are the percentages with respect to the number of 5-HT-immunoreactive cells in the subregion or combined subregions. The numbers in parentheses beneath the columns headed “Non-5-HT retrograde” are the percentages with respect to the sum of 5-HT and non-5-HT retrogradely labeled cells. Approximately twice as many sections were included in the counts for animal 138 as for animal 150.

167

KWIAT AND BASBAUM

-

AP 3.0

.4.


..

A

0

.

8i

5-HT project

.

.

.

.

mnon-5HT project

05-HT

FIGURE 7. Camera lucida drawingsof the rostroventral medulla made from individual3@pmsilver-enhanced,5-HT-immunoreacted sections. The distributions of 5-HT and non-5-HT-immunoreactivecells retrogradely labeled from the dorsal horn are shown in A. Triangles, double-labeled cells; squares, retrogradely labeled, non-5-HT-immunoreactive cells; open circles, 5-HTimmunoreactive cells, not retrogradely labeled. Ipsilateral to the injection site is on the left in each drawing. The method for dividing the rostroventral medulla into midline and lateral areas for purposes of cell counting is depicted in B. The A-P readings are taken from the atlas of Paxinos and Watson (1986).

DISCUSSION Since our injections were restricted to a single spinal segment, it is possible that the number of retrogradely labeled cells is a significant underestimate of the total number of brainstem neurons projecting to the dorsal horn of the spinal cord. Indeed, the rather low percentage 168

of midline NRM cells that were doubled-labeled suggests that considerable numbers of bulbospinal projection neurons were missed. This observation, however, reinforces our claim that the injection site did not result in uptake by large numbers of axons of passage. Thus, although our results may not be generalizable to all segments of the spinal cord, they demonstrate that the



FIGURE8. 5-HT- and non-5-HT-immunoreactiveretrogradely labeled cells in the paragigantocellularis (A) and raphe magnus (B)nuclei. Solid arrows,double-labeledcells;arrowheads, non-5-HT retrogradely labeled cells;open arrows: 5-HT-immunoreactive but not retrogradely labeled cells. Calibration bars: 10 pm.

brainstem origin of the noradrenergic innervation of the cervical dorsal horn in the rat is widespread and includes noradrenergic cells of the LC, SC, A5, and A7 cell groups. In terms of absolute numbers, no single population predominated. Our results do not support the conclusion of Fritschy and Grzanna (1990) that the LC is the predominant, if not exclusive, source of dorsal horn NA-containing terminals.

Furthermore, although most studies have focused on the midline raphe region as giving rise to a dorsally projecting serotonergic pathway, our results suggest that the majority of the dorsal-horn-projecting 5-HTimmunoreactive cells are located lateral to the midline, in the PGL and ventral part of Gi. Finally, confirming the results of Skagerberg and Bjorklund (1985), we also found that a large population of non-5-HT-immuno169

KWIAT AND BASBAUM

reactive cells in the PGL projects to the cervical dorsal horn.


Methodological Considerations

Since our previous experiments demonstrated that injections of WGA:apoHRP-Au into the lateral funiculus of the spinal cord produce little or no retrograde labeling in the brainstem (Basbaum and Mendtrey, 1987), the retrograde labeling resulting from injection of WGA:apoHRP-Au into the dorsal horn is likely to have arisen predominantly via uptake by axon terminals in the region of the injection. Differences in the number of retrogradely labeled cells found for the different injections probably resulted from the varying amounts of tracer that were deposited in the dorsal horn. The high viscosity of the WGA:apoHRP-Au and the compressibility of the fluid used to make the injection may have contributed to the dficulty of injecting exact tracer volumes. Of course, differences in the number of retrogradely labeled cells in a given region may also be attributed to the differences in the placement of the tracer in the dorsal horn. The pattern of retrograde labeling that we observed in the RVM is, in fact, consistent with the conclusions derived from our early anterograde tracing studies (Basbaum et al., 1978, 1986) and from studies that combined retrograde tracing with white matter lesions of the spinal cord (Martin et al., 1978; Basbaum and Fields, 1979; Skagerberg and Bjorklund, 1985). Specifically, it was demonstrated that the NRM and adjacent PGi and ventral Gi nuclei project primarily to the dorsal horn via the dorsolateral funiculus, whereas the more caudal raphe nuclei, obscurus and pallidus, project via the ventrolateral funiculus to ventral horn sites of termination. In fact, the primary reason for including an analysis of 5-HT-immunoreactive projection neurons of the RVM in the present study was to confirm these earlier findings and to provide supporting evidence that the WGA:apoHRP-Au tracer injections did not enter the ventral horn. Indeed, the injections were focused in the superficial laminae, and we found few labeled cells in raphe obscurus and pallidus. Finally, since the C1 cell group in the ventrolateral medulla projects to the IML (Ross et al., 1981), the absence of retrograde labeling in that region served, in all cases, as an internal control to rule out the possibility that the retrograde labeling pattern resulted from uptake by axons of passage that terminated in the IML. Since uptake of WGA:apoHRP-Au by cut axons is limited, we do not believe that the small amount of tracer deposited in the dorsal columns could account for the widespread retrograde labeling of cells in the brainstem. The dorsal columns consist mainly of as170

cending primary afferent collaterals, descending corticospinal fibers, and some axons originating in the dorsal column nuclei (Burton and Loewy, 1977). A few retrogradely labeled cells were, in fact, found in the dorsal column nuclei. These were presumably neurons with axons that projected to the dorsal horn. The fact that retrogradely labeled cells were found in the dorsolateral medulla, in a region that included the medial and lateral vestibular nuclei, was unexpected. Previous studies of vestibulospinal projections indicated that these nuclei project exclusively to ventral horn motor neuron pools. Most of these studies, however, have been done in the cat and monkey; anterograde tracing studies that would reveal the spinal terminations of vestibular nuclei have not been performed in the rat. TH-Immunoreactive Projecting Cells

The origin of the noradrenergic innervation of the spinal cord dorsal horn has been the subject of considerable debate. In part, the lack of agreement is a consequence of limitations of the methods used in earlier studies. Because it has been difficult to restrict injections to small areas of the spinal cord, and because of problems associated with uptake and transport by damaged axons and axons of passage, retrograde tracer studies have not provided definitive information with regard to innervation of specific regions of the spinal cord. With the exception of the recent study of Clark et al. (1991), conclusions about the origin of projections to specific spinal cord regions have relied heavily on data from anterograde autoradiographic tracer studies and from studies in which changes in spinal transmitter content were measured following lesions of selected brainstem catecholamine cells (for a review, see Westlund et al., 1982). The general consensus resulting from these studies was that the LC and SC project to all areas of the spinal cord, and that the A5 cell group is at the origin of a projection to the intermediolateral cell column. As described above, the generally held view that the LC projects predominantly to the ventral horn of the spinal cord has been questioned (Fritschy et al., 1987; Lyons et al., 1989; Fristchy and Grzanna, 1990). On the basis of PHA-L anterograde tracing and selective neurotoxic effects of DSP-4, these authors concluded that the LC projects primarily to the dorsal horn and intermediate regions, and only minimally (if at all) to the ventral horn. These authors also demonstrated that the A5 and A7 cell groups are at the origin of a ventrally projecting pathway that is distinct from the coeruleospinal pathway. They could not, however, rule out the possibility that these cell groups also project to the dorsal horn. Although the neurotoxin DSP-4 appears



to be selective for a subpopulation of spinally projecting NA axons, the selectivity is not based entirely on topographic organization of brainstem NA cells. In fact, there is a significant reduction of retrogradely labeled NA cells from all spinally projecting NA cell groups following DSP-4 treatment (Lyons et al., 1989). The fact that DSP-4 affects widely distributed NA populations suggests that the susceptible neurons, despite their spatial separation, share some property or feature that confers sensitivity to DSP-4. Our finding that the LC, SC,As,and A7 cell groups all project to the dorsal horn is consistent with lesion studies indicating that the noncoerulear populations contribute to the dorsal horn innervation (Nygren and OIsen, 1977; Clark and Proudfit, 1991b), and provides anatomical corroboration for the pharmacological and physiological evidence that multiple brainstem noradrenergic cell groups contribute to the descending regulation of spinal nociresponsive neurons. For example, electrical andor chemical stimulation of each of the noradrenergic cell populations shown in the present study to project to the dorsal horn produces behaviorally measured antinociceptive effects (Jones and Gebhart, 1986; Aimone et al., 1987; Janss et al., 1987; Burnett and Gebhart, 1988; Miller and Proudfit, 1990). In some cases the antinociception could be reversed by intrathecal administration of adrenergic antagonists, indicating that noradrenergic projections were involved. Our results suggest that the NA-mediated antinociceptive effects produced by electrical or chemical stimulation in either the LC, SC,AS, or A7, in fact, result from direct activation of a descending NA projection to the dorsal horn; an indirect activation of another NA descending projection from any of these regions, of course, cannot be ruled out. How descending noradrenergicprojections are activated by PAG and RVM stimulation is not understood. Anatomical tracing data have not implicated any one NA cell group in the spinal NA-mediated antinociceptive effects produced by stimulation in the PAG and RVM. In fact, anterograde and retrograde tracing studies have demonstrated that the PAG and RVM project to several brainstem NA cell groups, including the LC, SC, AS, and A7 (Sakai et al., 1977;Cederbaum and Aghajanian, 1978; Byrum and Guyenet, 1987; Clark and Proudfit, 1991a). There is not, however, complete agreement among investigators. Thus Aston-Jones et al. (1986) reported that the LC receives a restricted afferent input from only PGi and the nucleus prepositus hypoglossi of the rostral medulla. Although Clark and Proudfit (1991a) demonstrated a projection from the RVM to the region of the A5 cell group, Byrum and Guyenet (1987) did not report this projection. Since the region of PGi in the rat receives a projection from the PAG

(Gallagher and Pert, 1978; Beitz, 1982; Van Bockstaele et al., 1989), it is possible that bulbospinal LC neurons could be activated indirectly from the PAG, via a connection in the medulla. 5-HT-Immunoreactive Projecting Cells

Although the brainstem origin of spinally projecting 5HT- and non-5-HTcontaining neurons has been studied extensively (see Bowker et al., 1982, for a review; Skagerberg and Bjorklund, 1985; Mendtrey and Basbaum, 1987), there is disagreement as to the proportion of spinally projecting neurons in the RVM that are serotonergic. Bowker et al. (1981, 1983) reported that over 80% of the medullary raphe nuclei neurons retrogradely labeled following large spinal HRP injections were also 5-HT-immunoreactive. Skagerberg and Bjorklund (1989, using aldehyde-induced fluorescence to detect 5-HT, found more non-5-HT-immunoreactive than 5-HT-immunoreactive projecting cells in the RVM. More recently, Jones and Light (1990) injected PHAL into the RVM and found a large number of anterogradely labeled fibers and terminals in the dorsal and ventral horns that could not be double-labeled for 5HT immunoreactivity. Our results confirm that there is a large population of non-5-HT dorsal-horn-projecting cells interspersed among the 5-HT cells of the RVM and caudal ventral pons. In fact, non-5-HT projection cells outnumbered the 5-HTprojection cells. Although our study has focused on the projection to a single cervical segment, the results provide new information on the distribution of 5-HT neurons in the ventromedial region of the rostral medulla and caudal pons that project specifically to the dorsal horn. We conclude that the majority of dorsal-horn-projecting 5HT neurons in this region are located lateral to the midline NRM, in the lateral PGi and in the ventral part of Gi, ipsilateral to the dorsal horn injection. Although Skagerberg and Bjorklund (1985) studied the distribution of spinally projecting 5-HT neurons in the ventromedial medulla and caudal ventral pons following a restricted injection of the retrograde tracer true blue, they did not find such a differential distribution. This discrepancy may be related to the fact that different tracers were used; true blue is diliicult to restrict to small areas and is readily taken up by fibers of passage. Summary

The distribution of retrogradely labeled TH-immunoreactive cells described in this report provides strong evidence that the noradrenergic innervation of the Cs dorsal horn arises from widespread brainstem cell populations, including the LC, SC, As, and A7 cell groups. 171

KWIAT AND BASBAUM

These data support pharmacological and physiological evidence that has implicated these cell groups in the descending control of spinal cord nociresponsive neurons. Since many of the noradrenergic cell groups that we have demonstrated to be at the origin of dorsal horn projections receive inputs from diverse brain regions (perhaps with the exception of the LC; Aston-Jones et al., I%), our results provide evidence that the noradrenergic regulation of nociceptive transmission at the spinal cord level arises from direct spinal projections of several brainstem noradrenergic cell groups.


ACKNOWLEDGMENTS

We thank Dr.Jon Levine for help with writing the paper, and Ms. Margaret Mayes and Ms. Elene Valdivia for their help with histology and photomicrography. This work was supported by U.S. Public Health Service Grants No. NS 21445 and No. NS 14627.

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Modification of serotonergic innervation of the rat spinal cord dorsal horn after neonatal capsaicin treatment.

Modification of serotonergic immunoreactive pattern in the dorsal horn of the rat spinal cord following dorsal root rhizotomy.

Plasticity of the serotonergic innervation of the dorsal horn of the rat spinal cord following neonatal capsaicin treatment.

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Dynamic alterations in the cutaneous mechanoreceptive fields of dorsal horn neurons in the rat spinal cord.

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