Brain Research, 288 (1983) 33-48 33 Elsevier

Descending Serotonergic, Peptidergic and Cholinergic Pathways from the Raphe Nuclei: A Multiple Transmitter Complex

R. M. BOWKER 1,*, K. N. WESTLUND 1, M. C. SULLIVAN l, J. F. WILBER 2 and J. D. COULTER 1

J Marine Biomedical Institute and the Department of Physiology & Biophysics and Psychiatry & Behavioral Sciences, The University of Texas Medical Branch, 200 University Boulevard, Galveston, TX 77550-2772, and 2Department of Internal Medicine, Louisiana State

University Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112 (U.S.A.)

(Accepted April 12th, 1983)

Key words: serotonin - - substance P - - enkephalin - - TRH - - immunocytochemistry - - spinal cord - - raphe nuclei

The localization of serotonergic, various peptidergic and possibly cholinergic neurons in the medullary raphe nuclei that project to the lumbosacral spinal cord have been studied using a retrograde transport method combined with immunocytochemical and histo- chemical techniques. Spinally projecting neurons stained for serotonin-like, substance P-like, enkephalin-like and thyrotropin-releas- ing hormone-like immunoreactivity and for the histochemical marker acetylcholinesterase were all observed in each of the raphe nu- clei of the medulla, as well as in the adjacent ventrolateral reticular formation. The similar distributions of the descending serotonergic and peptidergic neurons in the raphe nuclei as well as quantitative data on their relative numbers suggest that a large fraction of raphe- spinal neurons contain serotonin co-existing with one or more peptides in the same cell.

INTRODUCTION

Numerous anatomical and biochemical studies have contributed information on the organization of

the brainstem raphe nuclei and their descending pro- jections to the spinal cord. The early histofluores- cence studies of Dahlstrom and Fuxe 23.24 identified

the presence of the indoleamine serotonin (5-HT) in

neurons of the raphe nuclei. In the medial brainstem,

beginning in the caudal medulla and extending

through the midbrain, the fluorescing cells of the raphe system were classified into 9 distinct cell groups numbered B 1 through B 9. Subsequently, others employing improved histofluorescent and histo- chemical techniques 9,16,21,26.28.31,42 and, most recent-

ly, immunocytochemical methods for serotonin 67,

have provided a wealth of data on the localization

and cellular morphology of the serotonergic neurons

of the raphe complex. The cells of the raphe nuclei which give rise to spi-

nal projections were first identified by retrograde chromatolysis following spinal injury15,69 and later by

retrograde transport of the enzyme horseradish per- oxidase (HRP) 2,6.20,22.46.47.51,70. From these studies,

the raphe spinal system is known to originate mainly

from the nucleus raphe obscurus, the nucleus raphe

pallidus and the nucleus raphe magnus, including lat- eral extensions into the adjacent ventral reticular for-

mation. These 3 medullary raphe nuclei correspond

approximately to the serotonergic cell groups B 1, B z and B 3 identified by Dahlstrom and Fuxe 23. As to

their spinal projections, anterograde autoradio-

graphic tracing techniques, as well as other degener-

ation methods, indicate widespread terminations by

raphe neurons in the spinal dorsal horn, particularly

the marginal zone, the ventral horn and the region around the central canal, at all spinal levels, and in

the intermediolateral cell column of the thoracic cord 5A°,34.49,5°. These termination zones correlate

well with the distribution of serotonin terminals in

the spinal cord 16,67. On the basis of these findings, it is

clear that the raphe spinal system has potentially

widespread and important roles in mediating de-

scending influences on spinal cord sensory, somatic motor, autonomic and reflex control functions.

It is widely assumed, though, that neurons project- ing to the spinal cord from the raphe nuclei employ serotonin as their neurotransmitter. The strongest

evidence comes from the early studies of Dahlstrom and Fuxe 2~ who observed swelling and chromatolytic

0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.

34

changes in certain indoleamine fluorescent cells of the medullary raphe nuclei following transection of the spinal cord. More recent studies which have com- bined various lesioning or tracing techniques with

histo- and immuno-chemical staining and biochemic- al assays for serotonin have supported this conclu- sion 7,49,58. Also, data obtained by the retrograde and

anterograde axoplasmic tracing methods correlate well with the distribution patterns of serotonergic cells in the brainstem 23,24.28,43.67, and serotonergic

terminals in the spinal cord 16,66. Electrical stimula-

tion in the medullary raphe has been shown to mimic

the effects of direct applications of serotonin on spi- nal cord neurons 3,19,32,35,54,63.71 .

While all of these findings support the conclusion that the raphe spinal system employs serotonin as

neurotransmitter agent, this may be an oversimpli- fied view. Various neuropeptides including sub- stances P, leucine and methionine-enkephalin, soma- tostatin and thyrotropin-releasing hormone have re- cently been localized in cells of the raphe nuclear complex 30,36,37,38. In some cases these peptides have

been found to co-exist in the same cells with seroto- nin 17,33.38,43. Studies have also indicated that many of

the neurons of the brainstem reticular formation, in-

cluding cells of the raphe nuclei, are likely to use ace- tylcholine as a neurotransmitter45, 60. These obser-

vations indicate the possibility that raphe-spinal neu- rons may contain transmitter substances other than serotonin. Newly developed methods which combine retrograde tracing and transmitter immunocyto- chemical techniques have, in fact, demonstrated that some raphe spinal neurons contain leucine and/or methionine enkephalin11,40 and substance pll.

Whether these neurons might also contain serotonin co-existing in the same cell has not been demon- strated anatomically, but biochemical data suggest that they do project to spinal cord 8,39,66. No detailed descriptions of the locations, numbers and cellular morphology of these peptide-containing raphe-spinal neurons are available, however.

For the studies reported here, a technique which combines immunocytochemical and histochemical staining methods for localizing serotonin, various peptides, and acetylcholinesterase, has been em- ployed to determine the putative transmitter sub- stances contained in raphe-spinal neurons. The ma- jor findings are that within the medullary raphe nu-

clei significant populations of neurons exist that con- tain substance P, leucine- and methionine-enkepha- lin and thyrotrophin-releasing hormone immuno- reactivity. A subpopulation of raphe-spinal cells was

found which also stained positively for

acetylcholinesterase. Comparing the relative num- bers of raphe-spinal neurons with positive staining for substance P with those stained for serotonin, it is apparent that a fairly large fraction (40%) of the raphe-spinal neurons contain both the monoamine serotonin and the peptide co-existing in the same

cell.

MATERIALS AND METHODS

In laboratory rats (200--400 g) anesthetized with sodium pentobarbital (25-35 mg/kg) a dorsal lami-

nectomy was performed at either the lumbar or cervi- cal enlargements and in some animals both spinal cord levels. A 25-50% solution (2-4/A) of horserad-

ish peroxidase (Miles) or a 0.1% solution (0.1-0.2/~1) of wheat germ agglutinin conjugated to HRP (WGA-HRP) was injected bilaterally into the spinal cord of different animals. Following a two-day sur- vival period, the animals were reanesthetized and perfused through the ascending aorta with 300 ml of warm (35-38 °C) physiological saline followed by 1 liter of cold (5-12 °C), 3.0-3.8% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). One liter of cold 30% buffered sucrose solution was infused after fixa- tion to prepare the brain and spinal cord for section-

ing. Tissue sections were cut at 20-25/am on a freezing

microtome and were placed in 0.1 M phosphate or 0.1 M Tris-HC! buffer (pH 7.2-7.4). The HRP histo- chemistry was enhanced by incubating the tissue sec- tions first in a 0.5% CoCI 2 solution prior to reacting them for the HRP reaction product. The tissue sec- tions were rinsed 2-3 times in Tris-HCl buffer, then reacted in a phosphate buffer solution containing 0.02% diaminobenzidine HCI and 0.01% H20 2 for 15 min. The retrogradely labeled cells containing HRP reaction product, when viewed in the micro- scope with bright field illumination, are seen to con- tain black punctate granules within the cytoplasm and extending into the proximal dendrites. A grada- tion of retrograde labeling in neurons is seen with some neurons being completely filled with blackened

35

reaction product while other cells may contain only a few HRP granules. Retrogradely labeled cells having light to intermediate amounts of HRP labeling were best suited for identifying double labeled cells stained by immunocytochemical methods.

The brainstem tissue sections were next incubated in antisera raised to serotonin (Immunonuclear Corp.) or to one of the peptides, substance P, leucine or methionine enkephalin, (all obtained from Im- munonuclear Corp.), and thyrotropin-releasing hor- mone (TRH) TM. Tissue sections were incubated at room temperature in the serotonin antiserum for 2 A, 48 h at dilutions ranging from 1:2000 to 1:8000. In experiments utilizing the various peptide antisera, it was necessary to treat the animals with the axonal transport inhibitor colchicine (Sigma), 24 h prior to perfusion in order to obtain immunoreactive staining of cell bodies for the various peptides in the brainstem. Forty-eight hours after the injection of HRP or W G A - H R P into the spinal cord, between 80 and 120/ag of a 1-2% colchicine solution was depos- ited intracerebrally. Subsequently, the brains and spinal cords were processed for HRP histochemistry as described above. The incubations of the tissues with the various antisera were at dilutions of 1:400-1:1200 for 2-3 days at room temperature.

After the antisera incubation, the immunocyto- chemical staining procedure followed the unlabeled antibody, peroxidase-antiperoxidase (PAP) method of Sternberger 68. Neurons stained positively for sero- tonin or peptide immunoreactivity contained rela- tively homogeneous light to dark brown coloration throughout the cell body including the dendrites.

In control experiments the various antisera were preabsorbed with an excess of their respective anti- gen for 24 h prior to incubation with the tissue sec- tions for 24-72 h 11.13. No positive immunoreactive staining was observed in tissue sections when the se- rotonin antisera was preabsorbed with 5-hydroxy- tryptamine (Sigma) at concentrations greater than 6.5/~M (2.5 mg per ml of the 1:1000 diluted antise- rum) or when the peptidergic antisera (diluted 1:400) were preabsorbed with their respective antigens (Beckman) at concentrations of 100 ~tg/ml.

On adjacent brainstem sections, histochemical staining for the enzyme acetylcholinesterase (ACHE) was performed using a protocol similar to that of Me- sulam 53. The HRP reacted tissue sections were first

washed extensively in distilled water and incubated in an acetylcholine medium at room temperature for 1-5 h. Ethopropazine (Sigma) was added to inhibit non-specific cholinesterase activity. After incubation in the acetylcholine medium the sections were rinsed extensively in distilled water and then reacted in 10% aqueous potassium ferricyanide (Sigma) solution for 2 min, washed in distilled water and routinely mounted and coverslipped.

Quantitative analysis of the relative numbers of raphe spinal neurons containing serotonin or sub- stance P immunoreactivity were carried out as pre- viously describedl2. Briefly, rats received large bilat- eral injections of HRP or W G A - H R P into the lum- bosacral spinal cord (2-5/tl of a 50% solution depos- ited over 12-15 spinal cord penetrations). This pro- cedure was done to retrogradely label as many spi- nally projecting neurons as possible. In the different brainstem 5-HT cell groups the numbers of 5-HT im- munoreactive neurons, HRP-filled cells, and double labeled neurons were counted in the same tissue sec- tions taken serially at representative levels through the brainstem. For each animal, the counts were con- verted to percentages. Since a number of factors, in- cluding tissue thickness, use of penetrating agents and the effective diffusion of colchine to block axonal transport, can affect the relative numbers of immu- nochemically labeled neurons (see ref. 13), data were only determined in brainstem sections having both optimal immunocytochemical staining and retro- grade labeling of neurons from the spinal cord.

RESULTS

Injections of HRP or W G A - H R P into the lumbar and/or cervical spinal cord followed by HRP histo- chemistry produced labeling in numerous spinally projecting neurons located within the medullary raphe nuclei and in the central parts of the reticular formation in the nucleus gigantocellularis (NGC). The retrogradely labeled cells in the raphe nuclei and NGC contained black punctate reaction granules dis- tributed throughout the cell cytoplasm and proximal dendrites. Large numbers of HRP labeled cells were found in the nucleus raphe obscurus (cell group B1), nucleus raphe pallidus (cell group B2) and nucleus raphe magnus (cell group B3). The distribution and cell morphology of the HRP filled neurons observed

36

A E

J

C D

Fig. 1. Photomicrographs illustrating serotonin immunoreactivity in raphe-spinal neurons. A: neurons from nucleus raphe magnus having serotonin-immunoreactive staining (open arrows) and serotonin staining and black HRP granules (filled arrows) after an injec- tion of HRP into lumbar spinal cord. B: low power photomicrograph shows nucleus raphe obscurus (RO) with serotonin- and double-

37

in the present study are in basic agreement with pre-

vious descriptions of reticulo- and raphe-spinal pro- jections labe led by the HRP method (refs. 6, 20,22,46,47,51,70) and need not be described fur-

ther. Retrogradely labeled neurons in the raphe nuclei

which contained positive immunoreactive staining for either serotonin or one of the peptides had black granules of HRP reaction product within a brown

stained cell cytoplasm. The brown coloration and generally homogeneous character of the immuno- chemical staining of the cytoplasm and dendrites of

these cells contrasted with the black, punctate HRP reaction product produced by the presence of HRP or W G A - H R P retrogradely transported from the

spinal cord. Thus, the double-labeled neurons were easily differentiated from neurons having only HRP granules or immunocytochemical staining. These 3 types of cellular labeling have been illustrated in col- or in a previous report 11. Examples in halftone pho-

tomicrographs are shown in Fig. 1.

Descending serotonin immunoreactive neurons Neurons containing serotonin immunoreactivity

were localized to the raphe nuclei and the adjacent ventral parts of the reticular formation in the medul- la. In the nucleus raphe obscurus (Fig. 1B) serotonin stained neurons were aligned bilaterally along the midline with the small to medium sized cells having

predominantly fusiform or multipolar shapes. Those spinally projecting serotonin immunoreactive neu- rons seen after a lumbar spinal cord injection of HRP were observed among neurons having only serotonin immunoreactive staining or retrograde labeling with HRP (Fig. 1C). Following injections of HRP in both the cervical and lumbar enlargements, most of the se-

rotonin stained neurons in the nucleus rapbe obscu- rus were observed to also contain HRP reaction product. However, a few spinally projecting neurons were consistently not stained for serotonin in this caudal raphe nucleus. Ventrally in the nucleus raphe

pallidus dense clusters of spinally projecting, seroto-

nin immunoreactive neurons were seen throughout the longitudinal extent of the nucleus and extending both laterally and ventrally to the pyramidal tract. Anterior to the inferior olivary complex in the nucle-

us raphe magnus and in the adjacent nucleus giganto- Cellularis, extending laterally from the midline, many

serotonin stained neurons were found to project to the lumbar spinal cord (Fig. 1D, 1E and 1F). In Fig. 1F, double labeled neurons can be seen in the nucleus gigantocellularis ventral to a group of reticuiospinal neurons (HRP labeling only). At the level of the tra- pezoid body a greater number of double labeled cells were observed after cervical spinal cord injections than after a lumbar spinal cord injection of HRP. In

the nucleus raphe pontis (cell group Bs) an occasional serotonin stained neuron was observed that was also retrogradely labeled from the spinal cord.

The large, multipolar shaped neurons found within

the raphe nuclei, which are characteristic of neurons in the reticular formation, consistently do not stain for serotonin immunoreactivity.

In Fig. 2 the distributions of serotonin-stained (5-HT) and double labeled (5-HT-HRP) neurons are shown following an injection of HRP into the lumbo- sacral spinal cord. Each dot represents one stained or double labeled cell as seen on the same 20/~m tissue section. The 5-HT stained, spinally projecting neu- rons are located, for the most part, in the nucleus raphe obscurus, nucleus raphe pallidus, nucleus raphe magnus and the adjacent ventral parts of the nucleus gigantocellularis.

In representative sections, the numbers of spinally projecting, serotonin stained neurons were counted and expressed as a percentage of the total number of spinally projecting neurons in these same raphe nu- clei. From this analysis 82.8% (665 of 803 neurons counted) of the HRP filled neurons were found to be stained for serotonin immunoreactivity. In addition, nearly all of the HRP filled cells in the nucleus raphe obscurus, raphe pallidus and raphe magnus and in the

labeled neurons. C: inset from B of RO illustrates two serotonin stained neurons (open arrows) and two double labeled cells (filled ar- rows). D: serotonin stained cell that projects to spinal cord is shown in nucleus raphe magnus. E: low power photomicrograph illus- trates nucleus raphe magnus (RM) and ventral parts of nucleus gigantocellularis of rat (P, pyramidal tract). F: high power view of dou- ble labeled (filled arrows) and large sized HRP filled neurons (stippled arrows) are shown. Serotonin antiserum was diluted 1:3000 in A and 1:4000 in B-F. Bars in A and C are 20 ~m and 25/~m, respectively, in D, 10/~m.

38

5HT 5HT'HRP

Fig. 2. Locations of serotonin (5-HT) immunoreactive and double labeled (5-HT-HRP) neurons in medulla of rat. The distributions of serotonin immunoreactive cells, as seen on a 22/~m section, are shown on the left while the 5-HT stained neurons that project to lum- bosacral spinal cord are plotted on the right from the same tissue section.

ventral parts of the nucleus gigantocellularis that did

not stain for serotonin immunoreact iv i ty were the

very large or giant sized mul t ipolar shaped neurons.

Many of these giant sized neurons which project to

the spinal cord do stain for the enzyme acetylcho-

l inesterase (ACHE) (see below).

Descending substance P immunoreactive neurons

In neuronal cell bodies , substance P immunoreac-

tivity is observed only after t rea tment of the animals

with the axonal t ranspor t inhibi tor colchicine. The

relative distr ibutions of the substance P positive cells

and the intensity of the staining varies somewhat

from animal to animal , apparent ly due to differences

in the effective area of diffusion of the colchicine.

The following descriptions of the substance P s tained

cells which project to the spinal cord were ob ta ined

from exper iments in which immunoreac t ive staining

was judged to be opt imal for substance P and the

other peptides. As with serotonin double labeled cells,

39

the spinally projecting neurons showing positive sub-

stance P immunoreactive staining had homogeneous- ly brown stained cytoplasm and contained black HRP reaction granules indicating the presence of retro- gradely transported HRP or W G A - H R P from the

spinal cord (Fig. 3B). The spinally projecting, substance P stained neu-

rons were located throughout the nucleus raphe ob- scurus, nucleus raphe pallidus and the nucleus raphe magnus and adjoining ventral parts of the nucleus gi- gantocellularis. In the nucleus raphe obscurus, sub- stance P immunoreactive neurons that projected to the spinal cord were predominantly small and medi- um sized cells with fusiform and multipolar shapes. The substance P -HRP filled cells were located as

two paramedial rows at the caudal portion of the nu- cleus raphe obscurus and coalesced into a single wide row anteriorly. The double labeled neurons were found among neurons having only substance P stain- ing or only retrogradely transported HRP granules. Ventrally in the nucleus raphe pallidus, numerous double labeled neurons were densely clustered in the region between the lobes of the inferior olivary com- plex. The oval shaped substance P stained neurons that projected to the lumbar spinal cord from this re- gion were slightly larger in size than those located dorsally in the nucleus raphe obscurus. This pattern

is similar to that observed for cells containing seroto-

nin immunoreactivity. Rostral to the inferior olivary complex, a large concentration of double labeled neurons was seen in the nucleus raphe magnus and

especially in the ventral parts of the nucleus giganto- cellularis. In the ventral parts of the nucleus giganto- cellularis, the substance P-HRP filled neurons were primarily fusiform or multipolar shaped (Fig. 3B).

Fig. 3B illustrates two double labeled neurons con- taining black HRP granules and brown homogeneous staining of the cytoplasm for substance P immuno- reactivity. The substance P double labeled neurons located in nucleus gigantocellularis extended lateral- ly from the nucleus raphe magnus, just dorsal to the

pyramids, toward the ventrolateral edge of the me- dulla to coalesce with a cluster of double labeled neu- rons as well as neurons stained only for substance P (Fig. 3A). At the level of the trapezoid body, the sub- stance P stained neurons did not appear to extend as far rostrally as the distribution of serotonin stained cells. Whether this difference in the rostrocaudal pat- terns of staining between the substance P immuno- reactive neurons and serotonin stained cells is due to incomplete staining for the neuropeptide in these re- gions must await further experimentation.

Fig. 4 illustrates the distribution of substance P-im- munoreactive (SUB P) and spinally projecting, sub-

B

Fig. 3. Photomicrographs illustrating substance P and double labeled neurons having both HRP granules and substance P-like staining (filled arrows) in medullary raphe nuclei of rat. A: low power photomicrograph of medulla anterior to inferior olive showing RP and ventral parts of reticular formation where many substance P and double labeled cells are located (P: pyramidal tract). B: the enclosed area in A shows two substance P immunoreactive neurons that project to lumbar spinal cord. Calibration bar in E is 25 ~m,

40

RM

NGC

SUB P SUB P- HRP

Fig. 4. Plottings showing substance P immunoreactive neurons (SUB P) on the left and double labeled (SUB P-HRP) from the same 22/~m sections on the right. The distributions of double labeled cells in nucleus raphe obscurus (RO), nucleus raphe pallidus (RP) and the nucleus raphe magnus (RM) including the ventral nucleus gigantocellularis (NGC) are similar to that of serotonin-stained spinally projecting neurons.

stance P stained neurons (SUB P-HRP) that were

found in the medullary raphe nuclei. The substance P stained and double labeled cells are plotted from the same 20-22 pm tissue sections. The localization of the substance P perikarya in the medulla following colchicine treatment are in agreement with the pre- vious observations of Ljungdahl et al. 48. The spinally

projecting, substance P stained neurons in the medul- la were confined to the 3 raphe nuclei and to the ven- tral parts of the nucleus gigantocellularis. The mor- phology and size of the substance P - H R P filled neu- rons in these 3 raphe nuclei was similar to that ob-

served for serotonin double labeled neurons. The percentage of raphe-spinal neurons that were

stained for substance P immunoreactivity was deter- mined in brainstem tissue sections having both opti-

mal immunocytochemical staining for substance P immunoreactivity and retrograde labeling of neurons with HRP from the lumbosacral spinal cord. The numbers of double labeled cells (SUB P-HRP) were counted and expressed as a percentage of the number of HRP filled neurons seen in the raphe nuclei and adjacent reticular formation on the same tissue sec- tions. The large sized, multipolar shaped cells lo- cated in the ventral parts of the nucleus gigantocellu-

laris, but dorsal to the substance P stained cells, were not included in the cell counts. Of the 1524 neurons that were observed to have HRP labeling in the raphe nuclei, 869 were found to be stained for sub-

stance P immunoreactivity. Thus 55% of the de- scending spinal cord projections emanating from the medullary raphe nuclei contain substance P-like im- munoreactivity.

Descending TRH and enkephalin-immunoreactive cells

Neurons immunoreactive to TRH were also seen to project to the spinal cord from the medullary raphe nuclei. These are considerably fewer in num- ber than those stained for substance P or serotonin. The TRH-stained spinally projecting neurons were concentrated in the nucleus raphe pallidus and in the ventrolateral part of the reticular formation. Nearly all TRH-stained neurons were found to project to the spinal cord. In the nucleus raphe pallidus, the double labeled neurons were oval shaped and were generally in the ventral parts of the nucleus. No TRH immuno- reactive neurons were seen more dorsally. Fig. 5A

41

shows the part of the nucleus raphe pallidus in which

TRH immunoreactive stained neurons were found to project to the spinal cord. Lateral to the inferior oli- vary nuclei, a second cluster of spinally projecting TRH-stained cells was located near the ventral sur- face of the medulla. In other regions of the medullary brainstem no TRH positive neurons were found. The

distributions of TRH stained neurons and double la- beled cells are plotted in Fig. 6.

Neurons stained for leucine- or methionine-enke- phalin were located in the nucleus raphe pallidus and the nucleus raphe magnus and the adjacent parts of the reticular formation. Anterior to the inferior oli-

vary complex, there is a major concentration of spi- nally projecting, enkephalin immunoreactive stained cells some of which are morphologically similar to those stained for serotonin and substance P. Fig. 5B shows two spinally projecting neurons located in the ventral nucleus gigantocellularis which are stained

for leucine-enkephalin.

Descending A ChE-containing neurons A population of spinally projecting neurons in the

medullary raphe nuclei and ventral nucleus giganto- cellularis exists which consistently do not stain with the serotonin or peptide antisera. These cells are very large, or giant sized, multipolar shaped neurons. This morphologically distinct group of neurons, which were characteristic of the nucleus gigantocel- lularis, can be localized throughout the medial third of the medulla, but were most numerous at brainstem levels between the anterior half of the inferior olive and the facial nucleus. These giant sized neurons were approximately 11/2 to 2 times larger in size than the serotonergic or substance P-immunoreactive neurons (Fig. 5C). An example of a giant sized neu- ron retrogradely labeled from the lumbar spinal

cord, is illustrated in Fig. 5C and 5D. In Fig. 5C, a low power photomicrograph of the nucleus raphe pallidus and nucleus raphe magnus and the adjacent ventral portions of the nucleus gigantocellularis (NGC) is shown. The serotonin-immunoreactive neurons (open arrows) and double labeled cells (filled arrows) are located in NGC and along the mid- line in the nucleus raphe magnus (NRM). The larger sized HRP filled neurons (stippled arrow), present among the 5-HT and double labeled neurons in these two nuclei, were not stained by the 5-HT antisera.

42

II,

t l i l t I I [

Fig. 5. Photomicrographs illustrating raphe-spinal neurons stained for TRH-tike (A) and leucine-enkephalin-like (B) immunoreactivi- ty and for the giant sized neurons (C and D) that stained for the enzyme acetylcholinesterase (E). A: illustration showing TRH immu- noreactive (open arrows), HRP filled (stippled arrow) and double labeled cells (filled arrows) in nucleus raphe pallidus (RP) of rat. B: two spinally projecting neurons in ventral nucleus gigantocellularis that are stained for methionine-enkephalin immunoreactivity. C: low power view of nucleus raphe magnus (RM) and nucleus gigantocellularis (NGC) with 5-HT stained cells (open arrows), double la- beled cells (filled arrows) and FIRP filled cells (stippled arrows). D: the inset illustrates an example of the large or giant sized neurons (stippled arrow) in RM that consistently do no t stain for serotonin. The large sized cell contains only HRP granules with a relative clear cytoplasm in contrast to the brown homogeneous staining of a 5-HT stained neuron with its long dendrite process (open arrow). E: large-sized cell in NGC stained for the enzyme acetylcholinesterase (filled arrow) that projects to lumbar spinal cord. Observe black granules in histochemically stained cell. Calibration bars in B, D and E are 25/~m (not corrected for tissue shrinkage).

43

NGC

SOt.

//

RV

NGC

TRH TRH - H R P

Fig. 6. Locations of TRH-like immunoreactive neurons and double labeled cells (TRH-HRP) in medulla as plotted from the same 22 /~m tissue sections.

44

Fig. 5D shows at a high magnification a giant neuron located in the nucleus raphe magnus which contains only black HRP granules indicating that it projects to the spinal cord. A serotonin-immunoreactive neuron with a long stained process can also be seen (open ar- row). Following staining for the enzyme acetylcho- linesterase, many of the giant cells in the raphe nuclei and adjacent nucleus gigantocellularis were double labeled (Fig. 5E). The small and medium sized cells in the raphe nuclei which projected to the spinal cord were no t stained for acetylcholinesterase.

DISCUSSION

The medullary raphe nuclei have long been viewed to be a collection of neurons which contain and utilize serotonin or a serotonin-like substance as their trans- mitter and/or neuromodulator. Both histofluores- cence21, 26,28,42 and immunocytochemica167 studies

have repeatedly documented this finding during the last two decades. As a result, many physiological and pharmacological studies on descending control have been oriented towards the presumed serotonergic influences mediated by these raphe cell groups upon various aspects of spinal cord function (cf. refs. 29, 52). For example, it is now well-known that stimula- tion of the nucleus raphe magnus and the adjacent re- ticular formation produces a profound inhibition upon the activity of interneurons 27,35,62,63,64 and as-

cending tract cells 72 that are excited by nociceptive responses. These effects are believed to be mediated by the serotonergic neurons projecting to the spinal cord from these medullary raphe nuclei. Further- more, serotonin inputs can produce an increased ex- citability of motoneurons 71 and monosynaptic and polysynaptic spinal cord reflexes TM, and also affect the autonomic preganglionic neurons of the thoracic spinal cord 1,19. These results, when taken together, along with numerous other reports, strongly impli- cate the serotonergic system originating from cell groups BI-B 3 as having important neuromodulator functions on various sensory, autonomic and motor systems of the spinal cord.

However, with the present demonstration that at least 5 different possible transmitters are contained within raphe neurons that project to the spinal cord, it is becoming increasingly evident that the raphe-spi- nal system is more complex than had been previously

thought. Studies attempting to determine the descend-

ing effects in the spinal cord mediated by these me- dullary serotonergic, peptidergic and cholinergic neurons have just begun to emerge as to their possi- ble neurotransmitter and/or neuromodulator actions. Of the peptides found to project to the spinal cord in the present study the undecapeptide substance P has, perhaps, received the most attention. Substance P has been shown to be released via a calcium depen- dent mechanism in a manner analogous to that de- scribed for established neurotransmitters4.39, 56,59. When applied locally to neurons in the dorsal or ven- tral horn of the spinal cord, substance P produces a slow but long-lasting subthreshold depolarization of the cell 59,73. As a result, spinal cord neurons become more excitable to any stimulus input, with the dis- charge rates of spontaneously active cells and of those activated by peripheral stimuli increasing dra- matically. Substance P is also believed to play an im- portant role in the activation of certain behaviors, such as locomotion and scratching 61.

The peptides leucine- or methionine-enkephalin and TRH have also been implicated as having a neu- romodulatory functions in the brain and spinal cord. Iontophoresis of the enkephalins near neurons in var- ious brain regions generally produces an inhibition of neuronal activity 14,25,57. For example, nociceptive re- sponses of thalamic neurons are depressed with local enkephalin application, while in the hippocampus, cellular activity may be enhanced. All enkephatiner- gic mediated effects are reversed by naloxone. TRH, when applied locally, produces a small long-lasting depolarization of spinal cord motoneurons similar to that of substance p55. The depolarization of the cell is

accompanied by an increased membrane conduc- tance and excitability of the cell. The presumed THR terminals mediating these effects in the spinal cord 41,44, in contrast to most other peptides, are de- rived exclusively from supraspinal sources. These ob- servations suggest in the spinal cord, that TRH mod- ulates the background activity of cells and facilitates transmission through more powerful synaptic inputs.

The co-existence of multiple transmitters in the same neuron has become an important issue in recent years as it challenges the concept that a given neuron contains and uses only one transmitter. All of the peptides in the raphe nuclei which have been demon- strated here to have descending spinal cord projec-

45

tions have been shown previously by otherst7, 33,38,39,43 to co-exist in neurons containing sero-

tonin. Neurons containing multiple transmitters have been demonstrated by a number of techniques, ei- ther employing two immunocytochemical proce- dures 3s,43, or combining immunocytochemistry and [3H]serotonin autoradiographyl7, 33. With these

methods, serotonin-containing neurons in cell groups

B1-B 3 have been shown to have positive immuno- reactivity for substance p17,38,43, TRH43 and enke- phalin 33.

The present analysis has provided an alternative

method to obtain information regarding the possible co-existence of multiple transmitters in single cells. This simple procedure not only identifies the efferent projections of a chemically defined population of neurons within a nuclear group, but also provides the basis for quantitative estimates of the relative contri-

butions of a particular cell group to a projecting sys- tem. Of the population of spinally projecting neurons localized in the raphe nuclei, nearly 85% were found to contain serotonin immunoreactivity t2,~3, while

55% of the raphe-spinal neurons were also stained positive for substance P-like immunoreactivity. These findings provide statistical evidence that near- ly 40% or more of the descending serotonin neurons which project to the lumbar spinal cord, also contain substance P. Thus, a considerable fraction of the de- scending raphe neurons appear to have multiple transmitters co-existing within the same neuron.

The significance of the existence of peptides and 'classical' transmitters in the same raphe-spinal neu- rons is presently not yet understood. However, the various functions of the descending raphe system may be better determined if the anatomical organiza-

tion of the serotonergic, peptidergic and cholinergic neurons within the raphe nuclei were known. One possibility is that the neurons comprising the raphe- spinal system consist of several distinct populations

which are subdivided on the basis of the single trans-

mitter or multiple transmitters that they contain. For example, the serotonergic neurons could be divided into neuronal subsets comprised of cells containing serotonin alone or one or more of the peptides. The latter subset could be further subdivided into addi-

tional subpopulations of cells which contain the enke- phalins, substance P, TRH or both. Each of these neuronal subsets with their different transmitter(s)

would potentially have different actions upon the spi- nal cord target cells.

Within the spinal cord, a number of different possi- bilities exist as to the functions of raphe-spinal neu-

rons containing single and multiple transmitter sub- stances. The released transmitter(s) might affect dif- ferent target cells or modify the activities of the same neurons in a differential fashion. In this regard, one transmitter might affect the postsynaptic neuron,

while the second transmitter might influence or mod- ulate an adjacent neuron, a presynaptic terminal that impinges upon the same post synaptic cell, or its own receptor located on the raphe-spinal nerve terminal itself 39. These numerous functional possibilities for

the raphe nuclei to interact with different target cells at the spinal cord level indicates that the raphe-spinal system potentially possesses an enormous complexi- ty. In the future, experiments designed to better un- derstand the many intricacies and variations in the organization of the raphe nuclei and their control on spinal cord functions will need to employ a variety of pharmacological approaches and functional tech-

niques.

ACKNOWLEDGEMENTS

We wish to acknowledge the typing assistance of Mrs. Debbie Pavlu and Mrs. Phyllis Waldrop. This study was supported by NIH Grants NS 12481 and

NS 11255.

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