Descending Noradrenergic Projections and their Spinal Terminations

Descending Noradrenergic Projections and their Spinal Terminations K.N. WESTLUND. R . M . BOWKER. M.G. ZIEGLER and J.D. COULTER Muririe Biomrdicul lristitutc, arid Depurtments o j Physioiogj rid Biophysics und P.s\;chitttr\; und Behaviorul Sciences. Uriiwrsiry of Texus Mctlicul Brunch. 200 University Boulevard. Galvcrtori, Te.r. 77550, and Uriii,crsity Hospitcd, Department of Medirine, Uiiivcrsiry of Cal$~rniu Mcdii.ul Cctiter, 225 West Dickinson Street, Sun Dicgo. C ~ i l ( fY2103 . (U.S.A.)

INTRODUCTION The noradrenergic innervation of the brain and spinal cord is derived from several distinct collections of catecholamine cells distributed from the rostra1 pons through the caudal medulla. The most conspicuous group of noradrenergic neurons lies within the nucleus locus coeruleus, although the other catecholamine cell groups (numbered A 1--A7 by Dahlstrom and Fuxe, 1964) contain noradrenergic neurons as well (Swanson and Hartman, 1975). While considerable attention has been given to the ascending noradrenergic projections, less is known of descending noradrenergic systems: Although Russell (1955) and Pickel et al. (1974) traced descending projections from the locus coeruleus in the pons as far caudally as the medulla, many different hypotheses about a coeruleospinal tract have been proposed (Papez, 1925 ; Dahlstrom and Fuxe, 1965). With regard to the descending noradrenergic terminations in the spinal cord. lesions in known noradrenergic cell groups, such as the locus coeruleus, are followed by the subsequent loss of spinal cord noradrenergic fluorescent labeling (Nygren and Olson, 1977). Other studies using the retrograde cell marker, horseradish peroxidase (HRP), show that spinal projections originate from cells in a number of brain stem regions known to contain noradrenergic cell bodies (Kuypers and Maisky. 1975; Hancock and Fougerousse, 1976; Crutcher et al., 1978; Basbaum and Fields, 1979 ;Coulter et al.. I979 : Martin et al.. 19791-3).These include the locus coeruleus (A6 cell group), the nucleus subcocruleus, the medial and lateral parabrachial nuclei and the nucleus of Kdliker-Fuse of the lateral pons (A7 cell group), neurons in the ventrolateral brain stem dorsal and lateral to the superior olivary and facial nuclei (A5 group), neurons located in the dorsomedial medulla around the nucleus of the solitary tract and dorsal motor nucleus of the vagus (A2 group), and neurons of the ventrolateral caudal medulla in the region of the lateral reticular nucleus (A1 group). While it would appear that the origins of descending noradrenergic projections may be quite extensive, an important issue is whether or not the cells in these nuclei which project to the spinal cord are in fact noradrenergic. Please address correspondence to: Joe Dan Coultcr. Ph.D., Marine Biomedical Institute, University of Texas Medical Branch. ZOO Univenity Boulevard, Galveston, Tex. 77550, U.S.A.

220 Previous attempts to determine the sources of catecholamine fibers in the spinal cord have used various histochemical staining methods (Satoh et al., 1977 ; Smolen et al., 1979) or have combined biochemical or histochemical methods with various types of lesions of catecholamine cells or their descending fibers (Dahlstrom and Fuxe, 1965; Kobayashi et al., 1974; Ross andReis, 1974; Nygren and Olson, 1977; Commissiong et al., 1978; Adkret al., 1979; Karoum et al., 1980). It is clear from these studies that all catecholamine terminals in the cord arise from supraspinal sources. However, there is far from unanimous agreement as to which cell groups give rise to these projections and to what extent a given cell group contributes to descending projections. The question of the precise origins and terniinations of descending noradrenergic projections to the spinal cord is an important one. These systems have potentially significant functions in regulating somatomotor, sensory and autonomic activity in the spinal cord (see, Westlund and Coulter, 1980). Physiological and pharmacological studies have implicated the nucleus locus coeruleus and subcoeruleus complex in central autonomic control, including regulation of respiratory and cardiovascular dynamics, micturition, and certain manifestations of affective behavior (e.g., defense reactions). Descending noradrenergic pathways may also be involved in sensory phenomena, including pain perception and analgesia. There is evidence that descending noradrenergic paths influence complex motor activities such as the central generation of locomotion and modulation of spinal reflexes. However, until it is firmly established which groups of noradrenergic cells project to the spinal cord, and on what groups of spinal neurons they terminate, the role of noradrenergic systems in all of these functions must remain largely speculation. In the experiments described here, two methods are used to identify noradrenergic (and possibly adrenergic) neurons which project to the spinal cord. One method involves the immunocytochemical localization of retrogradely transported antibody to dopamine-b-hydroxylase (DBH) following injection into the spinal cord. The second method, a double labeling technique, employs the retrograde transport of HRP to identify spinally projecting cells, combined with conventional DBH immunocytochemistry to simultaneously visualize DBH in these same cells. Using these two methods the precise origins of descending noradrenergic systems to the spinal cord can be determined. The results indicate that the major noradrenergic projections to the spinal cord arise from the various cell groups of the pons, including the nucleus locus coeruleus and the subjacent subcoeruleuslmedial parabrachial nuclear complex. However, the caudal medullary noradrenergic cell groups apparently do not have descending connections as previously believed. In view of the major contributions that the nucleus locus coeruleus and subcoeruleuslmedial parabrachial nuclei make to the descending noradrenergic system, their projections to the spinal grey matter have been examined in monkeys with the anterograde autoradiographic tracing method (Westlund and Coulter, 1980). The termination patterns, obtained autoradiographically , have then been compared with the pattern of noradrenergic fiber and terminal staining in the spinal cord localized immunohistochemically with the antibody to DBH. The patterns of spinal cord innervation revealed by these two methods are virtually identical suggesting that norepinephrine is the transmitter in both of these descending systems. ORIGINS OF NORADRENERGIC PROJECTIONS TO THE SPINAL CORD Our approach has been to identify the origins of descending noradrenergic terminations first using the retrograde transport of antibody to DBH. DBH antibody has been shown (Silver and

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Fig. I . Neurons of the ponh containing retrogradely transported antibody to dopamine-/%hydroxylase (DBH) from the spinal cord. A : low-power photomicrograph of retrogradely filled neurons of the Kdliker-Fuse nucleus. Fibers of the lateral lernniscus are located irnrnediately to the left of the labeled cells. B and C : higher magnification of DBH-labeled. multipolar neurons seen in enclosed area of A . Note dark reaction granules within the cytoplasm and proximal dendrites while the nucleus does not stain. I n C the labeled cell is approximately 23 ,urn in diameter. D: the caudal part o f the locus coeruleus nucleus showing retrogradely labeled cells in the ventral portion and scattered along the lateral edge 01 the nucleus. E: higher magnification of stained neurons in the ventral locus coeruleus showing the dense immunocytocheinical reaction product.

222 Jacobowitz, 1979 ; Pasquier et al., 1980) to be taken up and retrogradely transported centrally only by adrenergic systems. In these studies, DBH antibody injected into the spinal cord is axonally transported to known noradrenergic cell groups in the brain stem and localized immunocytochemically (Westlund et al., 1981). In labeled cells the immunocytochemical staining is similar in appearance to retrogradely transported HRP in that the cytoplasm and proximal dendrites of labeled neurons are filled with brown granular reaction product as well as diffuse lighter staining of the cytoplasm (Fig. 1B ,C). The nuclei are not stained. In contrast, after conventional DBH immunocytochemistry, the cytoplasm of immunoreactive cells contains a homogeneous brown staining. The retrogradely transported antibody (presumably still bound to DBH) thus appears to be in many small to medium-sized vesicles in the soma of labeled neurons, in contrast to endogenous DBH which is distributed throughout the entire cell cytoplasm. In control experiments the DBH antiserum is preabsorbed with an excess of DBH. Also, preimmune serum is injected into the spinal cord and the tissue sections processed as described previously. No positive staining is present in any of the control experiments. Those cells which are retrogradely labeled with the transported antibody to DBH from the spinal cord are located in the ventral part of the nucleus locus coeruleus (the A6 cell group of Dahlstrom and Fuxe, 1964), the nucleus subcoeruleus, the medial and lateral parabrachial nuclei and the Kolliker-Fuse nucleus (A7 cell group), and the region dorsal and lateral to the superior olivary nuclei (AS cell group). Cells retrogradely labeled with DBH antibody in the ventral nucleus locus coeruleus at the caudalmost extent of the nucleus are illustrated in Fig. ID,E. Labeled cells of the ventral locus coeruleus are medium-sized neurons with diameters [(length width)/2] ranging from I 1 pm to 23 pin. The cells are primarily multipolar and oval shaped, but fusiform cells are also present. Labeled neurons are concentrated at the caudal pole of the nucleus locus coeruleus, although a few labeled cells are seen in the ventral part of the nucleus throughout its extent. Fig. 2A shows the distribution of cells retrogradely labeled with DBH antibody following an injection of the antibody into the cervical spinal cord. Caudally, at the level just rostra1 to the

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Fig. 2. The distributions of retrogradely labeled neurons in the pons are plotted following an injection into the cervical enlargement of antibody to dopamine-/%hydroxylase in A and horseradish peroxidase (HRP) in B. Each plot shows single retrogradely filled neurons observed in a single 30 pin tissue section. A : the inmunoreactive DBH-filled cells are observed primarily in the ventral and caudal locus coeruleus (LC) although scattered labeled cells are seen throughout the rostrocaudal extent of the nucleus. Othcr arcas of cells containing the relrogrddely transported DBH antibody from the spinal cord include the subcoeruleus nucleus (SC), the medial and lateral parabrachial nuclei (MPB, LPB), the Kolliker-Fuse nucleus (KF) and the region dorsolateral to the superior olivary nuclei (SO). B : HRP-labeled neurons are localized in these same regions as well as other areas including the raphe complex (R), the vestibular nuclei (VN), and the pontine reticular formation (PRF). bc = brachiuni conjunctivum; bp = brachium pontis; CG= central grey; IC= inferior colliculus; LL= nucleus of the lateral lernniscus; Cun = nucleus cuneiformis; mlf= medial longitudinal fasciculus; MeV = mesencephalic nucleus and tract of the trigeminal nerve; MoV = motor nucleus of the trigeminal nerve; NTB = nucleus of the trapezoid body ; PG = pontine grey ; Pre = nucleus prepositus hypoglossi; PSV = principal sensory nucleus of the trigeniinal nerve ; SCN = superior central nucleus (Bechterew) ; tb = trapezoid body; Vent = IVth ventricle; V = tract of the spinal nuclcus of the trigeminal nerve.

223

B

A

DBH

HRP

224 genu of the facial nerve, perikarya containing retrogradely transported antibody are first seen in the ventrolateral central grey. Immediately rostral to this level (Fig. 1D,E), where the nucleus locus coeruleus assumes its characteristic crescent shape, the pattern of retrogradely labeled cells clearly delineates the ventral division of the nucleus. It is this ventral division of the locus coeruleus which makes a major contribution to the descending noradrenergic fibers projecting to the spinal cord. Labeled cells fill the narrow ventral portion of the nucleus but some are scattered along its lateral edge more dorsally. At this same level, the proximal dendrites and perikarya are aligned to accentuate the curved ventral and lateral borders of the nucleus. Rostrally, the perikarya are aligned most often in a medial-lateral direction with most dendrites having the same orientation. However, dendrites are seen extending beyond the borders of the nucleus in many directions. While the majority of labeled cells are within the confines of the ventral locus coeruleus some labeled cells lie in the adjacent central grey. Ventral to the locus coeruleus, in the subjacent nucleus subcoeruleus, another large populat i o n of neurons is retrogradely labeled with DBH antiserum. The subcoeruleus nucleus (A7 cell group) extcnds ventrolaterally from the locus coeruleus, through the pontine tegmentum in a diagonal band, to the ventrolateral border of the brain stem. Labeled cells and their processes are oriented with the long ventrolateral axes of the nucleus. Cell shapes and sizes are similar to those described for the ventral locus coeruleus, although fewer rounded, multipolar shaped cells are seen in the subcoeruleus nucleus. In the ventrolateral pontine tegmentum, the labeled cells of the subcoeruleus nucleus are continuous with retrogradely labeled cells of the A5 cell group caudally and with cells of the Kolliker-Fuse nucleus rostrally (A7 cell group). The labeled cells of the A5 cell group are distributed in an arc over the dorsolateral surface of the superior olivary complex. They are medium-sized, mostly fusiform in shape and ranging from 14 pm to 24 pm in size. In the rostral pons, the pattern of labeled cells extends laterally from the mid-region of the subcoeruleus nucleus to join the Kolliker-Fuse nucleus. Labeled neurons in the Kolliker-Fuse nucleus are slightly larger (18-25 pm), multipolar to oval-shaped cells with prominent nuclei (Fig. IA, B, C). Some of the labeled cells are scattered ventrolateral to the brachium conjunctivum and among fibers of the lateral lemniscus. The neurons labeled in this region lie in various orientations and have dendrites radiating in many directions. Within the medial and lateral parabrachial nuclei, neurons are seen which contain retrogradely transported antibody to DBH. These neurons are morphologically similar to the labeled cells of the subcoeruleus nucleus. When antibody injections are made into either the cervical, thoracic, or lumbosacral cord (20 experiments), the general pattern as well as the number of retrogradely labeled cells in the pons is virtually the same (Fig. 2A). This observation suggests that many of the descending noradrenergic fibers extend the length of the spinal cord. The relative numbers of retrogradely labeled cells, their distributions and their morphologies in each of the pontine noradrenergic cell groups are in close agreement with the patterns of labeled cells in these same groups following spinal cord injections of horseradish peroxidase (Fig. 2B). While HRP-labeled cells are located in other regions not containing noradrenergic cells, such as the vestibular nuclei, the raphe nuclear complex and the reticular formation. cells labeled with DBH antibody are located only in catecholamine cell groups A5, A6 and A7, areas known to contain noradrenergic neurons. This finding further substantiates previous observations that the retrograde transport of antibody to DBH is specific for noradrenergic (and possibly adrenergic) systems and that the antibody to the DBH enzyme is not transported by cells which do not contain the enzyme. In the caudal brain stem, no cells containing retrogradely transported DBH antibody are ever observed in the A1 group, located ventrolaterally in the medulla, or in the more dorsally

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Fig. 3. Photomicrographs illustrating HRP-labeled neurons from the spinal cord combined with staining by DBH immunocytochemistry. A : section through the mid pons showing the nucleus locus coeruleus stained for DBH immunoreactivity. At this level. only a few cells are retrogradely labeled with HRP making them easily visible among cells of the locus coeruleus which are stained with the antibody to DBH. B: higher magnification of the inset in A qhowinp cells of the ventral locus coeruleus nucleus labeled with both black granular HRP reaction product and DRH immunoreactivity (filled arrows). C : neurnns within the A I catecholamine cell group in the ventrolateral medulla which stain for DBH. Cells retrogradely filled with HRP (arrows) lie among the DBH-stained neurons. especially medially. No double labeled cells are seen in the cell groups A l and A2 in these studies. D: shows a neuron in the pontine subcoeruleus nucleus (A7 cell group) double labeled for DBH immunoeytochemistry and HRP transported froin the lumbar spinal cord. Note both homogenous DBH staining and punetate HRP granules in the cytoplasm and in dendrites. Bar: SO pm.

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situated A2 cell group. The absence of retrograde labeling of the A 1 and A2 cell groups from the spinal cord is not due to an inability to transport the DBH antibody, since injections of the antibody into the hypothalamus labeled large numbers of neurons in both cell groups. Apparently, these caudal medullary noradrenergic cell groups give rise to ascending projections rather than descending spinal pathways as previously supposed. Since medullary noradrenergic cell groups do not appear to project to the spinal cord, the pontine noradrenergic cells provide virtually the entire noradrenergic innervation of the spinal cord. The origins of descending noradrenergic pathways demonstrated by the retrograde transport of antibody to DBH have been confirmed using a second method that combines retrograde HRP localization of spinally projecting cells and conventional DBH immunocytochemistry (Bowker et al., 1981) to identify noradrenergic cell bodies. Neurons containing retrogradely transported HRP from the spinal cord are visualized first using CoCI2 followed by reaction with the chromagen 3,3'-diaminobenzidine hydrochloride. Retrogradely labeled cells contain a black punctate HRP reaction product within a relatively clear cytoplasm. In the same tissue sections, localization of DBH with conventional inimunocytochemical staining yields a homogenous brown staining of the cytoplasm. Neurons containing both markers have black punctate reaction product within a homogenous brown stained cytoplasm (Fig. 3D). DBH-labeled spinally projecting cells are found throughout the locus coeruleus, the subcoeruleus, the parabrachial and the Kolliker-Fuse nuclei, as well as around the superior olivary complex in the AS cell group. Again spinally projecting noradrenergic neurons are seen caudally in the ventral locus coeruleus nucleus (Fig. 3A and B). While cells of the dorsal locus coeruleus stain for DBH, only neurons in the ventral locus coeruleus are labeled both for DBH and as well as HRP retrogradely transported from the spinal cord. Fig. 3D illustrates a subcoeruleus cell containing black horseradish peroxidase granules transported from the spinal cord within the homogenous brown stained cytoplasm indicating immunoreactivity to the DBH antibody. The relationship between DBH positive cells and HRP-labeled spinally projecting cells is best illustrated using plots of representative sections through the brain stem (Fig. 4). Cells stained for DBH are plotted from individual tissue sections in Fig. 4A. DBH immunoreactive neurons are found within catecholamine cell groups : ( I ) Al , ventrolaterally in the medulla adjacent to the nucleus retroambiguus; (2) A2, dorsally in the medulla among cells of the dorsal motor nucleus of X and the nucleus of the solitary tract; (3) A4, along the lateral edge of the ventricle in the caudal pons; (4) A5, adjacent to the superior olivary complex and the facial nucleus; ( 5 ) A6, the locus coeruleus; and (6) A7 cells of the subcoeruleus, medial and lateral parabrachial and Kolliker-Fuse nuclei. These findings are in close agreement with the report by Swanson and Hartman (1975) on the distributions of DBH immunoreactive cells in the rat. HRP-labeled cells from the same sections are plotted in column B of Fig. 4. The distribution of cells labeled following HRP injections in the cord agrees with previous studies identifying spinally projecting neurons in the brain stem (Kuypers and Maisky, 1975; Hancock and Fougerousse, 1976; Crutcher et al., 1978; Basbaum and Fields, 1979; Coulter et al., 1979; Martin et al., 1979b). Cells containing both the retrogradely transported HRP from the spinal cord and immunohistochemically stained with DBH in the same sections are plotted in column C of the figure. These double-labeled cells include neurons located in the AS cell group around the superior olive, the A6 cell group of the ventral nucleus locus coeruleus, and scattered neurons distributed throughout the A7 cell group including the nucleus subcoeruleus, the medial and lateral parabrachial nucleus, and Kolliker-Fuse nucleus. Double-labeled cells are found only

227

DBH

HRP

DBH-HRP

Fig. 4. The locations of DBH immunorcactive neurons (A), cells retrogradely labeled with HRP from the spinal cord (B), and double-labeled neurons (C). are plotted from the same sections in the pons and medulla. Double-labeled neurons arc seen anion2 DBH-stained cells ofthc puns. hut not in the medulla. Abbreviations are the same as in Fig. 2. XI1 = hypoplossal nucleus; N R A = iiuclcus retroanibiguus; SpV = spinal nucleus of V .

as far caudally as the AS cell group and no double neurons are present in the A1 or A2 cell groups. The locations and pattern of double labeling of neurons in the pons agree with the findings obtained with the retrograde transport of the DBH antibody. In the medulla, DRH-labeled cells of the A I cell group are found at the ventrolateral edge of the reticular formation and scattered more medially (Fig. 3C). DBH-labeled cells are also observed dorsally associated with the dorsal motor nucleus of the vagus and nucleus of the solitary tract in the A2 cell group. After spinal cord injections HRP-labeled cells of similar morphology are observed with the A I and A2 cell groups of the medulla. However, in no case, did we find any neurons containing both HRP granules and DBH immunoreactivity in these medullary cell groups. Our studies involving two different techniques, ( 1 ) retrograde transport of DBH and (2) the retrograde transport of HRP in combination with DBH immunocytocheniistry, provide iden-

228 DESCENDING NA PATHWAYS

Fig. 5 . Schematized summary of the origins of descending noradrenergic pathways shown in sagittal view, Noradrenergic cells within the A 1 and A2 catecholamine cell groups of the medulla do not project to the spinal cord. Pontine catecholamine cell groups AS-A7 send descending projections to all spinal levels. Both the A 1 and A2 cell groups, as well as pontine groups AS-A7 project rostrally. Nomenclature based on Dahlstrom and Fuxe, 1964.

tical results. Together these studies support the view that pontine noradrenergic cell bodies are the major source of spinal noradrenergic terminals and that medullary noradrenergic groups do not project to the spinal cord. Noradrenergic neurons within the pontine cell groups of the locus coeruleus (A6), the A7 cell group of the subcoeruleus, parabrachial, and Kolliker-Fuse nuclei and the A5 cell groups give rise to descending noradrenergic projections to all spinal cord levels, including to the sacral cord. While medullary cell groups A1 and A2 do not appear to contribute a noradrenergic input to the spinal cord, noradrenergic neurons of these two medullary cell groups have ascending projections at least to the diencephalon. These findings are schematically summarized in Fig. 5 . TERMINATIONS OF DESCENDING NORADRENERGIC PROJECTIONS TO THE SPINAL CORD In light of the major contribution that the locus coeruleus and subcoeruleuslmedial parabrachial nuclei make to descending noradrenergic paths, the spinal projections from these two regions have been examined in monkeys with the anterograde autoradiographic tracing method (Westlund and Coulter, 1980). One of the major findings of these studies is the demonstration that the locus coeruleus projects to the region of the parasympathetic preganglionic neurons of the sacral spinal cord, while the subcoeruleuslmedial parabrachial nuclei project to the region of the sympathetic intermediolateral cell column of the thoracic cord. Axons from both the locus coeruleus and subcoeruleuslmedial parabrachial nuclei have terminations in the dorsal and ventral spinal grey matter, and within the region around the central canal at all spinal cord levels. The pattern of terminations demonstrated by autoradiography is remarkably similar to thc pattern of noradrenergic fibers and terminals in the spinal cord shown with DBH immunoq tochemical staining (Westlund and Coultcr, 1980). 1 he monkey appears to be a more suitable model for these studies than the rat since in the rat there appears to be a non-noradrenergic projection from the nucleus tegmentalis laterodorsalis (TLD) which lies adjacent to the nucleus locus coeruleus. The projections of the TLD nucleus are thought to be separate from the descending locus coeruleus pathway to the sacral spinal

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Fig. 6. Injection sites in the brain stem of 'H-labeled amino acids used to trace descending pathways with anterograde transpon autoradiography. Following long e x p o w e times (5 months), injections of isotope into the locus corrulrus ( A ) and the subcoeruleusimedial parabrachial (B) nuclei are schematically illustrated. Heavy stipplinp indicates the core of the injection site. while surrounding fine stipple indicates the maximal spread of injected amino acids. Stipple and dots indicate terminals and fibers respectively seen in the pontine tegmentum. (From Westlund and Coulter, 1980.)

cord (Loewy et al., I979b). In the monkey, no cell group in the vicinity of the locus coeruleus has been identified on cytoarchitectural grounds as being equivalent to the TLD nucleus of the rat. Further. based on HRP studies in the monkey, no spinally projecting neurons are located in the dorsolaterdl pons other than those in the locus coeruleus and subcoeruleusimedial parabrachial nuclei proper (cf., Westlund and Coulter, 1980). Iiijections of isotope into either the region of the locus coeruleus (Fig. 6A) or the subcoeruleusimedial parabrachial nuclei (Fig. 6B) labeled bundles of fibers which can be traced through all levels of the spinal cord with the autoradiographic technique (see Fig. 7). The descending coeruleospinal pathway is situated mainly ventrolaterally in the ipsilateral white matter of the cord with fibers extending into the dorsal part of the lateral funiculus to innervate the dorsal horn. Fibers descending from the region of subcoeruleusimedial parabrachial nuclei include a major component located ipsilaterally in the ventrolateral quadrant of the spinal cord, as well as a sinall contralateral component located dorsally in the lateral funiculus. In addition, fibers from both the locus coeruleus and subcocruleusimedial parabrachial nuclei are seen crossing the midline at all spinal cord levels. Terniinal labeling in the spinal grey matter, following isotopic injections into either the locus coeruleus or subcoeruleusimedial parabrachial regions, is heaviest over the ventral horn in the region equivalent to Rexed's ( 1954) laminae VII-IX, particularly over presumptive motoneurons (see Fig. 7). Lighter label appears around the central canal, Rexed's lamina X , in the dorsal horn marginal layer (laminal) and extending into the superficial part of the substantia pelatinosa (equivalent to Rexed's laminae 11). Labeling is also present in the spinal intermediate zone corresponding to Rexed's laminae IV-VI. Beyond these similarities, two different patterns of labeling appear depending on whether isotope injections are placed into the locus coeruleus or ventrally in the subcoeruleusimedial parabrachial region. A major descending projection can be traced from the locus coemleus to sacral spinal cord levels Sz-S4 where bilateral zones of terminal labeling are found in the parasympathetic preganglionic cell column. In contrast, descending axons from the subcoeruleusimedial parabrachial nuclei terminate most heavily at cord levels T,T4 in the region of the interniediolateral cell column, bilaterally.

230

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Fig. 7. Schematic illustration of spinal cord noradrenergic terminations localized using DBH immunocytochemistry (DBH) compared with autoradiographic localization of descending spinal terminations following isotope injections into the nucleus locus coeruleus ('H-LC) and the subcoeruleus/inedial parabrachial regions (3H-SC), The patterns of DBH immunoreactive terminals are plotted from sections of the spinal cord taken from the levels indicated. DBH-stained fibers and varicosities are localized in the superficial dorsal horn, in the ventral horn, around the central canal and in the thoracic and sacral autonomic preganglionic nuclei. The distribution of anterograde axonal (large dots) and terminal labeling (fine stipple) are plotted from autoradiographs of representative sections at the same spinal segmental levels. The labeling pattern from injection of isotope into the left locus coeruleus (LC) shows heaviest concentrations of silver grains bilaterally over the sacral cord segments (ST&) in the lateral cell column with less silver grain accumulations in the superficial dorsal horn, the interniediate zone. the ventral horn, and around the central canal. The labeling pattern from injections of isotope into the left subcoeruleusimedial parabrachial nuclei (SCIPB) shows heaviest accumulations of silver grains bilaterally in the thoracic cord segments (TTT~),as well as in the superficial dorsal horn, the ventral horn and around the central canal. Laminar borders are drawn according to Rexed (1954). (Modified from Westlund and Coulter, 1980.)

The pattern of autoradiographic labeling in the spinal cord corresponds closely to the pattern of noradrenergic terminals demonstrated by DBH immunocytochemistry . Fig. 7 compares the pattern of noradrenergic fibers localized using DBH immunocytochemistry with the pattern of descending locus coeruleus and subcoeruleus/medial parabrachial fibers and terminals in the spinal cord demonstrated autoradiographically. DBH immunoreactive fibers and terminal varicosities are concentrated in the ventral horn particularly around the large motoneurons (Fig. 8B), around the central canal, and in the dorsal horn marginal layer extending into the

23 I

Fig. 8. Photomicrographs of DBH imtnunocytocheiiiically stained terminals in the spinal cord. A : within the dorsal fibers and tcrminal varicosities from a fine meshwork with radiating horn of the sacral spinal cord. Ini~nun~ireactive fibers extending through Inmina I 1 into lamina 111. Note that the immunoreactive fibers both extend parallel to the laminae as well as radiate perpendicularly to them in the dorsal horn. Stained varicosities are especially dense in the lateral cell column (lower centcr). B : the ventral horn shows very dense staining of DBH immunoreactive terminal varicositieb cspccially at the lunibar arid cervical enlargements. I n this example the terminals are seen to surround ;I presuiiiptive niotoneuron.

substantia gclatinosa at all cord levels (Fig. 8A). Staining is particularly intense i n the intetinediolateral cell column of the thoracic cord (Fig. 9A,B) and in the corresponding region of the lateral cell column of the sacral spinal cord (Fig. 9C,D). The localization of DBH immunoreactivity in the sacral spinal cord has only recently been described by our laboratory (Westlund and Coulter, 1980). Thus, there appears to be a differential distribution of noradrenergic terminals in the spinal cord depending on whether the projections arc traced from the region of the locus coeruleus located dorsally in the pons, or from the subcoeruleuslmedial parabrachial regions located more ventrally. These new observations are schematically summarized in Fig. 10. The descending projections from the locus coeritleus are observed to terminate heavily among the parasympathetic preganglionic cells of the sacral cord and in the region of the dorsal motor nucleus of the vagus i n the brain stem. In contrast, the descending subcoeruleuslmedial parabrachial terminations are heaviest within the region of the sympathetic preganglionic cells of the thoracic cord. I n addition, axons from both of these noradrenergic groups of the pons terminate throughout the spinal grey especially in the marginal layer of the dorsal horn, around the central canal. and among the motoneurons of the ventral grey.

Locus cocnrleus ptithways The present stitdies confinn previous work in the rat, cat, monkey and opossum (Kuypers and Maisky. 1975; Hancock and Fourgerousse, 1976; Crutcher et al., 1978; Basbaum and Fields, 1979: Coulter et al., 1979; Martin et al., 1979b) using retrograde and anterograde axonal tracing methods which have identified descending spinal projections from the locus coeruleus. The use of inimunocytochemical techniques, involving axonal transport of antibody to DBH and double labeling with conventional DBH immunocytochemistry, significantly extend previous observations by showing that the cells of origin of the descending locus coeruleus pathway contain DBH, the synthesizing enzyme for norepinephrine. This is the best

232

Fig. 9. The pattern of DBH immunoreactive terminals at thoracic and sacral spinal cord levels. A : sections from thoracic cord levels T r T 4 show the pattern of dense varicosities associated with the intermediolateral cell column, extending toward the midline, and around the central canal. B : inset in A at higher magnification showing staining around the central canal (CC) and the intermediolateral cell column (IML). C: sections from the sacral cord, (S2-S4) illustrate intense DBH immunoreactivity in the dorsal horn, ventral horn, around the central canal, and in the lateral cell column. D : at higher magnification the inset in C of the lateral autonomic preganglionic cell column shows DBH terminal varicosities surrounding unstained cell bodies. Laterally the white matter remains clear.

evidence to date that these spinally projecting locus coeruleus neurons employ norepinephrine as a neurotransmitter. The conclusion that the locus coeruleus is one of the major areas providing noradrenergic innervation of the spinal cord is also in accord with other histochemical and biochemical studies (Kobayashi et al., 1974; Satoh et al., 1977; Aditr et al., 1979; Smolen et al., 1979; Karoum et al., 1980). Previous studies (Dahlstrom and Fuxe, 1965; Ross and Reis, 1974; Nygren and Olson, 1977 ; Commissiong et al., 1978) combining various histochemical staining methods and lesions of descending noradrenergic fibers indicate that the locus coeruleus contributes descending fibers to the spinal cord ventral horn, to the dorsal horn (mainly the superficial part) and to the region around the central canal, but does not have major terminations in the intermediolateral cell column of the thoracic cord. These observations are consistent with the present findings and with studies using the autoradiographic tracing techniques (Commissiong etal., 1978;Holstegeetal., 1979; Loewyetal., 1979a;Martinetal., 1979a,b;Goodeetal.,

233

Descending NA Pathways Locus Coeruleus

Subcoeruleus

all levels

T2-4

'2-4

@

P

Fig. 10. Schematic summary of the niajor descending pathways to the spinal cord from the locus coeruleus and the subcoeruleus nuclei. The locus coeruleus projects heavily to regions concerned with parasympathetic autonomic control including the dorsal motor nucleus of the vagus ( X ) and the sacral cord intermediolateral cell column (IML) bilaterally. The subcoeruleus projections descend to innervate regions controlling sympathetic autonomic function, including the thoracic cord intermediolateral cell column, and somatic cranial nerve nucleus of the hypoglossus (XII).

1980: Westlund and Coulter, 1980). Unfortunately, in none of the previous histochemical studies were the effects of locus coeruleus lesions on the noradrenergic innervation of the sacral cord examined, nor have studies employing the anterograde autographic tracing technique documented projections from the locus coeruleus as far caudally as the sacral cord. Otherwise the findings of previous studies are largely in agreement with the observations here concerning the spinal terminations of the descending projections of the locus coeruleus. The conclusion that the locus coeruleus is the origin of descending noradrenergic projections to the sacral spinal cord, conflicts with recent studies in the rat (Loewy et al., 1979b). As noted earlier, in the rat a small group of cells lying near the nucleus locus coeruleus, termed the nucleus tegmentalis laterodorsalis (TLD), is thought to give rise to descending projections to the sacral cord. Based upon anterograde and retrograde tracing studies combined with 6-hydroxydopamine to destroy noradrenergic cells of the locus coeruleus, it has been concluded that projections to the sacral cord arise from the TLD nucleus, but not the locus coeruleus, and that the descending TLD pathway is non-noradrenergic. That the sacral cord is, in fact,

234 innervated by noradrenergic fibers is demonstrated by the present studies in rat which show heavy concentrations of DBH immunoreactive terminals in the sacral spinal segments, especially in the region of the lateral parasympathetic cell column. That the locus coeruleus is, at least in part, the origin of the DBH immunoreactive terminals in the sacral cord is suggested by the large numbers of locus coeruleus neurons which retrogradely transport the DBH antibody from injection sites in the sacral spinal segments. Together these findings confirm earlier evidence (Westlund and Coulter, 1980) that the locus coeruleus has major descending noradrenergic terminations in the sacral spinal cord. In regard to the descending TLD pathway, preliminary data from our laboratory indicate that many cells within this nucleus that project to the spinal cord contain substance P-like immunoreactivity . These observations suggest that two parallel pathways exist to the sacral spinal cord from the dorsolateral pons : one originating from the locus coeruleus which is noradrenergic, and a second one arising from the TLD and which may be peptidergic. Projections of the locus coeruleus to the region of the sacral preganglionic parasympathetic cell column, as well as to the region of the dorsal motor nucleus of the vagus and the Edinger-Westphal nucleus of the brain stem, suggest involvement in the regulation and coordination of central parasympathetic outflow (Westlund and Coulter, 1980). This is consistent with previous physiological studies which have implicated vairous regions of the dorsolateral pons, including the locus coeruleus, as participating in central autonomic control. Electrical stimulation of the nucleus locus coeruleus evokes widespread changes in cardiovascular dynamics (Ward and Gunn, 1976). Other studies have been concerned with spinal projections from the region of the locus coeruleus which are involved in micturition (Barrington, 1914, 1925; Kuru and Yamamoto, 1964; Kuru, 1965), although the direct participation of the descending locus coeruleus pathway in micturition is unclear. In addition to innervating various autonomic cell groups, the locus coeruleus projects extensively and bilaterally to somatic sensory and motor cranial nerve nuclei and to the spinal dorsal and ventral horns. The terminations of axons from the locus coeruleus in the superficial laminae of the dorsal horn are consistent with the postulated role of descending noradrenergic pathways in modulating sensory transmission (Engberg and Ryall, 1966; Segal and Sandberg, 1977; Belcher and Ryall, 1978; Reddy and Yaksh, 1980), in particular the transmission of nociceptive information. With regard to motor functions, the proximity of the locus coeruleus to sites where electrical stimulation evokes spinal locomotor activity has been noted along with pharmacological evidence indicating an important role of descending catecholamine pathways in the generation of locomotor behavior (Grillner, 1975). Subcoeruleus p u t h w q

The noradrenergic neurons of the nucleus subcoeruleus, along with the adjacent parabrachial nuclei and the Kolliker-Fuse nucleus have been considered together as comprising the A7 cell group originally defined by Dahlstrom and Fuxe (1964). Based on the pattern of termination in the brain stem and spinal cord, the subcoeruleusimedial parabrachial nuclei clearly give rise to a pathway distinct from the descending locus coeruleus system. Further investigations are required to determine whether the other descending noradrenergic neurons of the A7 group including cells of the lateral parabrachial nuclei, the nucleus of Kolliker-Fuse, as well as neurons scattered in the ventrolateral brain stem extending caudally to the A5 group around the superior olivary complex should be included as part of the “subcoeruleus” descending system. As zhown by the numbers of DBH immunoreactive neurons with descending spinal projec-

225 tions, neurons in the subcoeruleus/medialparabrachial nuclear complex provide a major part of the descending noradrenergic innervation of the spinal cord. Their spinal terminations differ from those of the nucleus locus coeruleus, however, in that a heavy projection exists to the thoracic intennediolateral cell column. Only sparse terminations exist in the brain stem and spinal cord regions giving rise to parasympathetic outflow, such as the dorsal motor nucleus of the vagus and the sacral spinal cord. This observation contrasts with the heavy noradrenergic innervation of these structures by the descending locus coeruleus pathway. The pattern of terminations of the subcoeruleus/medial parabrachial pathway suggests that the descending noradrenergic projections from these brain stem regions are involved in central control of sympathetic outflow. This conclusion is in accord with previous studies indicating that the thoracic intennediolateral sympathetic cell column receives a dense noradrenergic innervation of supraspinal origin (Dahlstriim and Fuxe, 1965 ; Glazer and Ross, 1980) and that neurons of the dorsolaterdl pons in the region of the subcoeruleus complex innervate the intermediolateral cell column of the thoracic cord (McBride and Sutin, 1976; Holstege et al., 1979; Loewy et al., 1979a,b; Martin et al., 1979a,b; Saperand Loewy, 1980). Furthermore, electrophysiological studies employing electrical stimulation in the subcoeruleuslmedial parabrachial nuclei indicate a role for this brain stem region in cardiovascular regulation (Wang and Ranson, 1939), in respiratory control (Bertrand and Hugelin, 197 I ) , and in the mediation of defense reactions (Coote et al., 1973). In contrast to the projections to autonomic prcganglionic cell groups, the projections of the subcoeruleus system to the spinal dorsal horn, the region of the central canal and in the ventral horn are similar to those of the descending locus coeruleus pathway. This finding indicates that both pathways share in some functions related to somatic sensory, motor and certain autonomic activities.

Other descending noruclrenergic paths Compared to the descending pathways originating in the locus coeruleus and subcoeruleusi medial parabrachial nuclei, the descending spinal projections from the region of the A5 noradrenergic cell group appear to be relatively modest. From more caudal medullary noradrenergic cell groups including the A1 and A2 regions and other scattered noradrenalinecontaining cells, spinal projections are non-existent. Previous studies (Loewy et al., 1979a.b) have identified direct spinal projections from the AS cell group to the thoracic intermediolateral cell column. In this regard, the A5 group appears similar to the descending subcoeruleusiinedial parabrachial nuclei pathway and might be considered as a caudal extension of the “subcoeruleus” descending noradrenergic system. The lack of descending noradrenergic projections from other A1 and A2 catecholamine cell groups of the caudal medulla is somewhat unexpected since cells in both these regions do have descending spinal terminations (Kuypers and Maisky, 1975 ; Crutcher et al., 1978 ; Basbaum and Fields, 1979; Coulter et al., 1979; Martin et al., 1979b) and a number of earlier observations (Satoh et al., 1977 ;Smolen et al., 1979) suggested that descending noradrenergic projections arose from these two cell groups. However, neither of the techniques used in the present study for identifying descending noradrenergic pathways show spinal projections from noradrenergic neurons of the caudal medulla. This observation cannot be accounted for by an inability of these cells to retrogradely transport the DBH antibody since injection of the antibody into the hypothalamus and other diencephalic sites labels many neurons in both the A I and A2 regions. Furthermore, it has recently been shown using these techniques that some

236 of the noradrenergic cells in the caudal medulla contribute to the vagus nerve (Ritchie et al., 198I ) . The conclusion from these studies is that, contrary to earlier evidence, the noradrenergic neurons of the A1 and A2 cell groups do not project to the spinal cord. Thus, with the exception of a few cells located in the rostra1 medulla in the vicinity of the facial nucleus, which probably belong to the A5 group, virtually all of the noradrenergic innervation of the spinal cord is derived from the pons. In the pons, the locus coeruleus and the subcoeruleuslmedial parabrachial nuclei make, by far, the largest contributions to the descending noradrenergic projections to the spinal cord. SUMMARY The origins and terminations of descending noradrenergic projections to the spinal cord are described using anterograde and retrograde axonal transport techniques combined with dopamine-P-hydroxylase (DBH) immunocytochemistry. The retrograde transport of the antibody to DBH, as well as a double labeling method employing the retrograde transport of HRP combined with DBH immunocytochemistry, are used to map the origins of descending noradrenergic pathways to the spinal cord. Spinal cord projections are found to arise from noradrenergic cells of the ventral locus coeruleus, the nucleus subcoeruleus, the medial and lateral parabrachial nuclei, the nucleus of Kolliker-Fuse, and the region around the superior olivary nuclei. Medullary noradrenergic cell groups do not appear to contribute projections to the spinal cord. Anterograde autoradiographic tracing studies indicate that the locus coeruleus projects to regions giving rise to preganglionic parasympathetic outflow, namely, the dorsal motor nucleus of the vagus and the sacral intermediolateral cell column. The subcoeruleusl medial parabrachial nuclei have descending projections to the region of the preganglionic sympathetic motoneurons of the thoracic intermediolateral cell column. Both the locus coeruleus pathway and the subcoeruleuslmedial parabrachial system project, at all spinal cord levels, to the dorsal horn (mainly the superficial part), to the region around the central canal and to the ventral horn in the vicinity of the large motor neurons. The patterns of termination of these two descending systems agree remarkably with the pattern of noradrenergic terminals in the spinal cord as defined by DBH immunocytochemistry. Together these studies indicate that the locus coeruleus and subcoeruleuslmedial parabrachial systems have major roles in mediating descending noradrenergic influences on spinal sensory, motor and autonomic functions. NOTE ADDED IN PROOF Since submission of this manuscript Ross et al. (Nrurosci. Lett., 25: 257--262) have demonstrated epinephrine cells in the ventrolateral medulla which project axons to the spinal cord, confirming our conclusion that spinally projecting neurons observed in previous studies using HRP histochemistry andor catecholamine histofluorescence techniques contain amines other than norepinephrine. ACKNOWLEDGEMENTS The authors wish to express their sincere appreciation to Ms. Merry Sullivan and Dr. Barbara Bowker for expert technical and photographic assistance. Also we thank Mrs. Susan

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