Afferents to brain stem nuclei (brain stem raphe, nucleus reticularis pontis caudalis and nucleus gigantocellularis) in the rat as demonstrated by microiontophoretically applied horseradish peroxidase

Brain Research, 144 (1978) 257-275 © Elsevier/North-HollandBiomedicalPress

257

AFFERENTS TO BRAIN STEM NUCLEI (BRAIN STEM RAPHE, NUCLEUS RETICULARIS PONTIS CAUDALIS AND NUCLEUS GIGANTOCELLULARIS) IN THE RAT AS DEMONSTRATED BY MICROIONTOPHORETICALLY APPLIED HORSERADISH PEROXIDASE

DOROTHY W. GALLAGER* and AGU PERT Section on Biochemistry, Biological Psychiatry Branch, National Institute of Mental Heath, Bethesda, Md. 20014 (U.S.A.)

(Accepted July 29th, 1977)

SUMMARY Using a retrograde tracer technique with microiontophoretically applied horseradish peroxidase (HRP), afferent projections to the brain stem raphe nuclei (BR, raphe magnus, pallidus and obscurus) and to two adjacent reticular nuclei, nucleus reticularis pontis caudalis (nRPC) and nucleus gigantocellularis (nGC) were identified. The most striking difference between the afferent projections to the BR and the adjacent nuclei as determined by this method is that afferents to the BR originate primarily from structures rostral to the pons, especially the mesencephalic central gray and the dorsal and ventral tegmentum. In contrast, the two reticular nuclei studied (nGC and nRPC) received afferent projections within or caudal to the ponsmedulla. For example, the nGC receives prominent afferent projections from the gray matter of the spinal cord. In addition, evidence for interconnections between all of the adjacent nuclei (BR, nGC and nRPC) was found. Such afferent projections are compatible with the notion that the brain stem raphe nuclei may serve as connections within the brain stem for a descending system, while the nGC may be a relay in a feedback loop between the spinal cord and the reticular formation.

INTRODUCTION The serotonergic pathways in the CNS have their cell bodies located primarily in the mesencephalic and brain stem raphe nucleilL The mesencephalic raphe nuclei appear to be the origin of an ascending serotonergic system4,1~, while the brain stem * Visiting Scientist (IPA: NTE 080778) on leave from Department of Psychiatry,Yale University School of Medicine, New Haven, Conn. 06508, U.S.A.

258 nuclei appear to have descending serotonergic projections to the spinal cord la. Recent anatomical studies employing either autoradiographic6,1a, 4s or horseradish peroxidase (HRP) retrograde transport techniques2, 38 have described afferent and efferent projections of the mesencephalic (dorsal and median) raphe nuclei. However, less information is available concerning the connections of the brain stem raphe system. Studies determining the efferent connections of these nuclear groups by retrograde degeneration 7, histochemical fluorescence la and most recently by autoradiography ~ all suggest a prominent connection to the spinal cord. At present only a few afferent projections to these nuclei have been described including the cerebral cortexS, 6°, the fastigial nucleus of the cerebellum s, the ventrolateral funiculus of the spinal cord 8 and the mesencephalic central gray a2. Precise identification of inputs to these nuclei is important, since recent evidence has implicated a descending serotonergic pathway in the mediation of reflexive behavior 49 as well as opiate antinociception 5,49. In the present study, we have attempted to identify the afferent projections of the brain stem raphe nuclei using the horseradish peroxidase (HRP) retrograde transport technique. This method has been used to determine the cells of origin of monoamine nuclear groups~,9,as, 53, and has several advantages compared to lesiondegeneration techniquesZa,zg,31,4°, 41,5°. In addition, in order to minimize tissue damage and to confine the enzyme to these small nuclei, delivery of HRP was accomplished using electrophoresis through micropipettesZ, iv. We have also examined the afferem projections of adjacent reticular nuclei (nucleus reticularis pontis caudalis (nRPC) and nucleus gigantocellularis (nGC)) using these techniques. METHODS

Preparation of animals and mieroiontophoresis Twenty-five male albino rats (Sprague-Dawley/ARS) weighing 220-300 g were used in this study. Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic instrument. Microiontophoresis of H R P was accomplished by the technique of Graybiel and D e v o d v as modified by Aghajanian and Wang 2. Micropipettes (2 mm diameter glass tubing loaded with a few strands of fiberglass prior to pulling) were prepared to have a tip diameter of 20~,0 #m. Pipettes were filled by capillary action following a direct injection into the tubing of a 25 ~ solution of H R P (Worthington Biochemical) in 0.01 M sodium chloride. Impedance of these micropipettes ranged from 3 to 10 M ~ when measured at 60 Hz through 0.9 ~ sodium chloride. In most animals a burr hole was drilled in the skull directly above the intended ejection site using the coordinates BR (P, 2.0 ram, L, 0 mm, H, --8.0 to --8.5 mm, n = 10); nGC (P, 3.0 mm, L, 1.0 mm, H, --8.0 to --8.5 mm, n = 4); nRPC (P, 1.5 ram, L, 1.0 mm, H, --8.0 mm, n -- 4). In some rats (n = 5) electrodes were positioned at an angle of 30° to vertical. This oblique approach avoided some areas passed through with vertical electrode placement. In two rats, ejections into the BR area were made with micropipettes filled only with 0.01 M sodium chloride. Micropipettes were lowered into the various reticular nuclei using an hydraulic microdrive assembly. Two/~A of positive current were applied to the HRP solution for 2 min by means of a high-voltage constant current source.

259

Determination of the quantity of HRP ejected In order to determine the amount of HRP which is ejected from the micropipettes under these conditions, some micropipettes were filled with tritiated HRP. Micropipettes with similar tip diameters and electrical impedances to those used to eject HRP into experimental animals were filled with a 25 ~o solution of [3H]HRP (New England Nuclear) in 0.01 M sodium chloride. Radioactivity of a 0.1 ml saline solution was measured following the ejection of the tritiated HRP for 2 min with 2 #A of positive current. Histochemical reaction for HRP The HRP histochemical technique used is a modification of the original technique described by Graham and Karnovsky 16. Sixteen to 20 h after the microiontophoretic ejection of HRP, the animal was anesthetized and the brain fixed by intracardiac perfusion with a solution consisting of 1 ~o paraformaldehyde and 1 ~o glutaraldehyde in 0.05 M phosphate buffer (pH 7.3). The brain was then removed and rinsed in 4 °C Tris buffer 0.05 M, (pH 7.6). Frozen transverse serial sections (40/~m) of the brain were cut and collected into ice-cold 0.05 M Tris buffer (pH 7.6). Every fourth section was soaked in an ice-cold preincubation solution containing a freshly filtered 1.4 mM solution of 3,3-diaminobenzidine (DAB) (Baker Chem.) in 0.05 M Tris buffer (pH 7.6) for 0.5 h. After this period, 0.03 ~ hydrogen peroxide was added to the DAB solution reaching a concentration of 1 mM hydrogen peroxide. Sections were gently agitated in this solution for 1.5 h, and then washed and mounted. Some sections were lightly counterstained with cresyl violet. Each section was examined for HRPcontaining cells under both light- and dark-field illumination. RESULTS In brain tissue, the dense core of the HRP reaction product resulting from the microiontophoretic application of HRP had diameters ranging from 0.3 to 0.8 mm. In the nGC and the nRPC, these ejections were nearly spherical in shape;however, in the BR area, the ejections were commonly eliptical, approximately conforming to the contours at these midline nuclei (Fig. 1). As reported in other studies~,9,17, the microiontophoretic ejection of HRP resulted in minimal grossly observable tissue damage. However, under dark-field illumination, cells adjacent to the ejection site showed diffuse, poorly differentiated reaction product, which may represent incomplete incorporation into cytoplasmic vesicles 40 or local uptake by cell bodies and dendrites of unmyelinated fibers2, 3a. In addition, some fibers surrounding the ejection site were observed to have a uniform brown coloration, which according to some investigators a°,40 is characteristic for an axonal reaction secondary to injury. However, in these studies, only cells which displayed a distinct stippling with HRP reaction product and multiple varicosities25,30,40,41 were considered as HRP-positive cells. When measured in vitro under our experimental conditions, it was determined that 2.2 -~ 0.3 #g of HRP is ejected (n = 10 electrodes, in vitro conditions described in Methods). However, in agreement with recent reportsa4, as, very small, discretely

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Fig. 1. Bright-field photomicrograph of an HRP ejection into the brain stem raphe (BR) nuclei. The genu of the facial nerve (GF), the motor nucleus of the trigeminal nerve (V), the nucleus gigantocellularis (GC) and nucleus reticularis pontis caudalis (RPC) can also be seen on this section. Diameter of dense core of HRP deposit is 0.4 mm. Section is 40 t~m thick and counterstained with cresyl violet. Scale bar = 0.5 mm.

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263 localized injections of HRP often failed to result in appreciable retrograde labeling. In our study, we found that if less than approximately 1.5 #g (as measured in vitro) was ejected into the BR area, no HRP-positive cells could be found outside the immediate ejection site. This lower limit of HRP necessary for transport from the brain stem raphe area may not be uniform throughout brain tissue, however, since a cell's capacity to accumulate HRP may be related to the number and size of its terminals ~1, diffusion barriers 41 or other unknown factors. Criteria as described by Nauta et al. 41 were used to differentiate labeled cells from endothelial cells. In addition, no HRP-positive-appearing cells were detected in animals (n = 2) in which 0.01 N NaCI (without HRP) was iontophoresed into the BR region and tissue sections were incubated as described in Methods. This suggests that endogenously present material was not being confused with HRP-labeled neurons a7,41. Possibly due to the small amounts of HRP iontophoresed and/or the highly localized ejection site, no more than 6-8 HRP-positive cells were found in a single tissue section. However, labeled sites were highly reproducible from animal to animal.

(1) Location of afferents to the brain stem raphe nuclei As shown in Fig. 1, ejection of HRP resulted in a densely stained central 'core' (0.4 ram) surrounded by a more lightly stained zone. Lateral borders of this area are poorly defined and the staining of lateral processes appears to support the suggestion that in some places the raphe nuclei fuse with the adjoining reticular formation 55. The limit of a typical ejection site within the BR is shown diagrammatically in Fig. 2. This site corresponds to the coordinate (P 2000) according to the atlas of AbadAlegria 1. The location of HRP-positive neurons resulting from this ejection is also illustrated in Fig. 2. In Table I, the distribution of BR afferents (n ---- 10 rats) as detected by the HRP method is compared with the distribution of nRPC (n = 5 rats) and nGC afferents (n = 5 rats). Following ejections into the BR area, HRP-positive cells were identified in the mesencephalic central gray (CG), especially the dorsal and ventrolateral areas excluding the region of the dorsal raphe nucleus (DR). Although, in all 10 animals, approximately 25 ~o of labeled cells were located in the CG area (Fig. 3), only two positive cells were identified in the DR area. HRP-positive cells were most numerous in the dorsal tegmentum (37 ~o of all labeled cells). Labeled cells were also found in the ventral tegmentum, and the deeper layers of the superior colliculus. In two animals where very anterior sections were obtained, a few HRPpositive cells were found in the ventromedial prefrontal cortex az. Although diffuse reaction product made examination of regions bordering the ejection site more difficult, HRP-positive cells were identified in the nRPO, nRPC, VIIIM and the nGC. However, with ejections of HRP confined to the BR area, approximately two-thirds of HRP-labeled cells were found within or rostral to the midbrain region. No HRPpositive cells were observed in the cerebellum. Similar labeling with HRP was observed when an oblique approach (30 ° to vertical) was used to position the micropipette (n ---- 2). (2) Location of afferents to the nRPC Iontophoresis of HRP into the nRPC (n = 5) produced an area of intense reac-

264

Fig. 3. Dark-field illumination photomicrograph of a mesencephalic central gray (CG) cell showing retrograde transport of H R P following an ejection of H R P into the BR region. Portion of ventral tegmentum (VT) also visible. Scale bar -- 20/~m.

265

Fig. 4. Bright-field photomicrograph of an H R P ejection into the n. reticularis pontis caudalis (RPC). Dark material lining electrode tract above facial nerve (F) is hemolyzed blood while material below F is H R P deposit (0.3 m m diameter). Other structures identified on this section are the n. reticularis pontis oralis (RPO), and the motor nucleus of the trigeminal nerve (V). Section is 40/~m thick and counterstained with cresyl violet. Scale bar = 0.5 mm.

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Fig. 6. Bright-field photomicrograph of an H R P ejection by an oblique approach into the nucleus reticularis gigantocellularis (GC). The medial vestibular nucleus (VIIIM), the nucleus praepositus hypoglossi (XIIP) the medial longitudinal fasculi (FLM) and pyramidal fibers (FP) can also be seen in this section. Diameter of core of H R P deposit is 0.7 mm. Section is 40/zm thick and counterstained with cresyl violet. Scale bar = 0.5 mm.

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tion product as illustrated in Fig. 4. The distribution of HRP-labeled neurons is shown in Fig. 5 and Table I. In contrast to afferents of the BR, nRPC afferents rostral to the hindbrain region accounted for only approximately 30 ~ of HRP-positive cells. Afferents to the nRPC as demonstrated by the H R P retrograde transport method include in the mesencephalon: dorsal tegmentum, ventral tegmentum and deep layers of the superior colliculus. Only 2 ~ of the H R P cells were found in the CG. In the metencephalon, HRP-positive cells were located in the medial vestibular nucleus, nRPO, with extensive labeling of the contralateral nRPC and nGC. HRP-positive cells were also located in the BR. However, as reported in other studies 26,53, the proximity of a structure to its ejection site makes ipsilateral localization of cells in some areas impossible. As in the case of the BR, when the micropipette was introduced into

269 i

Fig. 8. A: dark-field photomicrograph of HRP-positive cells in the ventrolateral gray horn (VH)of the cervical spinal cord following an ejection of HRP into the nGC. Lateral funiculus (LF) contains no reactive cells. B: no reactive cells are seen in the ventrolateral horn following an ejection of HRP into either the BR (this section) or nRPC (not shown). Scale bar ~ 5/~m. the n R P C by an oblique approach (n = 1), labeling was found to be similar to the vertical placement, although a few cells were found in the posterior hypothalamic area, only by this approach.

(3) Location of afferents to the nGC As illustrated in Fig. 6, iontophoresis of H R P into the n G C resulted in an area of intense reaction product approximately 0.7 m m in diameter (n = 5). The distribution of HRP-labeled neurons is shown in Table I and illustrated in Fig. 7. Again, in contrast to BR afferents, afferents to the n G C which are located rostral to the hindbrain region account for only 25 ~ of HRP-positive cells. Afferents to the n G C as demonstrated by this method include, in the mesencephalon: dorsal and ventral tegmentum, occasional labeling of C G and deep layers of the superior colliculus. In the metencephalon, HRP-positive cells were located in the nRPO, VIIIM, contralateral nRPC, n C G and the BR. As in the case of the n R P C ejections, ipsilateral positive cells in the n R P C and n G C could not be clearly identified following ejections in the nGC. The most numerous reactive cells were located in both the dorsal and ventral gray of the cervical spinal cord (Fig. 8). Labeling here appeared to be exclusive to the nGC, since no HRP-positive cells were found in the S.C. following ejections in either the BR or the nRPC. When micropipettes were lowered into the n G C area at an angle

270 30 ° to vertical (n ---- 2), labeling was found to be similar to the vertical placement, although a few cells were found in the posterior hypothalamic area using this approach. DISCUSSION Notes on the technique Afferents from the brain stem nuclei including the BR, the nRPC and the nGC have been demonstrated in several studiesS, 36,53,5~. However, with the exception of the nRPC 53, the limitations of the degeneration and autoradiographic techniques used to demonstrate these afferent projections have made it impossible to identify precisely which cells project to these nuclei. The H R P retrograde transport method has many advantages compared to lesion-degeneration techniques which have been well documented by others 2s,al,4°,41,5°. In addition, by applying H R P microiontophoretically, which results in a highly concentrated, well-localized deposit of the enzyme, some of the problems associated with the H R P retrograde transport method have been reduced 2,9, 17. In this study we have tried to control for artifactual labeling by using both oblique and vertical micropipette placements and to control for individual variation by using several experimental animals. However, since the uptake of H R P 38,41 and other proteins ~4 may not be uniform among neurons within a nucleus, using the retrograde transport technique one cannot assume that lack of H R P labeling is equivalent to the absence of an afferent projection. Because of this and other limitations of the H R P technique 2a,2s,33,41, we have compared our findings when possible to published studies of these regions using other anatomical and physiological techniques. The most striking finding in this study is that there are major differences between the afferent projections of the BR and adjacent reticular nuclei. While approximately two-thirds of HRP-positive cells following BR ejections were found anterior to the pons, only one-fourth of the cells were found labeled rostral to this region following ejections into the nRPC or nGC. This suggests that (1) diffusion into the adjacent nuclei after the microiontophoretic ejection of H R P was not prominent and (2) although there is some overlap between the projection areas of these nuclei, at least quantitatively the afferent projections to the raphe nuclei can be distinguished from the afferents to the nRPC and nGC. As demonstrated by the HRP retrograde transport method, an afferent projection almost unique to the BR was found in the dorsal and lateral mesencephalic central gray area, excluding the region of the dorsal raphe nucleus (DR). Such an afferent projection has been suggested by both degeneration ~5 and autoradiographic ~2 studies. The failure to find any afferent connection from the DR area to the BR has also been observed in an autoradiographic study by Conrad et al. 11. However, in contrast to our study and the study by Conrad et al. 11 in the rat, Pierce et al. 48 have found autoradiographic evidence for a descending connection from the D R to the BR in the cat. Although the apparent discrepancy is not understood at this time, in our study several HRP-positive cells were localized around the borders of the D R region, suggesting that diffusion of [3H]proline to these adjacent cells could account for some of the BR labeling found in the autoradiographic studies 48. Both Conrad et al. 11 and Pierce et al. 4s using [3H]proline and Ha-

271 milton and Skultety18 using degeneration techniques have observed a diffuse projection to the nGC and nRPC and adjacent reticular formation nuclei from the DR area. Since we found no HRP-positive cells in the DR and only minimal (2-3 ~) labeling of the CG after ejection of HRP into the nGC and nRPC, it is possible that there may be a subpopulation of afferent cells from the DR and/or CG which do not take up HRP, at least in amounts sufficient for light microscopic detection41. In support of this possibility Sakai et al. 5a also failed to routinely observe HRP-labeled cells in the DR following injections of HRP into the nRPC, while a low density of HRP-positive cells were observed in the rest of the mesencephalic central gray. It is also possible that the projections to the nGC and nRPC observed using autoradiographic techniques is due to diffusion of the label from the CG, since Conrad et al. it observed that the density of autoradiographic grains to the reticular nuclei increased when [aH]proline was injected into the adjacent ventral tegmentum rather than directly into the CG. Another afferent projection which was positive exclusively for the BR in our study was the prefrontal cortex. This is consistent with studies in which degeneration of midline pontine nuclei in the rat a2 and medullary nuclei in the cat a were found following ablation of the prefrontal cortex. Following lesions of the 'motor area' of the cortex of the rat, Valverde 60 also found degeneration in the BR. However, in contrast to the abovementioned studies and our own, he found the contralateral reticular nuclei (nGC and nRPC) also to be affected. Although the BR, nRPC and nGC send efferent projections to the spinal cord as evidenced by degenerationT,42,57 and autoradiographic5 studies, only the nGC was found in our study to have an afferent projection from the cervical spinal cord (dorsal gray - - deep to lamina V, lateral and ventral gray). This pattern is consistent with a spinoreticular projection which has been demonstrated both anatomicallya6,39,51 and physiologicallyl0,15, 58. Several researchers have suggested that the spinoreticular pathway may relay nociceptive information from deep structures and high threshold cutaneous receptorsl0,15. However, recently Siegel and McGinty54 have reported that these reticular cells may be more concerned with motor function than nociception in the unanesthetized animal. Thus, the physiological significance of this pathway does not yet appear to be understood. One striking finding in our study was the appearance of HRP-labeled cells in the reticular formation contralateral to the injected area. These results are consistent with the autoradiographic studies of Walberg61 in which the injection of [aH]leucine in the left nGC and nucleus reticularis parvicellularis resulted in the localization of autoradiographic grains over cell bodies in the right nGC and nucleus parvicellularis. Walberg notes heavily labeled fibers crossing at the raphe. In our studies following ejection of HRP into the nGC or nRPC, some BR cells also exhibited the granular HRP reaction product indicative of retrograde transport from terminal areas. These observations suggest that adjacent as well as contralateral nuclei may be interconnected. In this regard, Sakai et al. 5a found HRP-positive cells in the nGC, nRPC and BR following injection of HRP into the nRPC. However, the HRP studies must be interpreted with caution, since uptake and retrograde transport of HRP by unmyelinated

272 axons from the midbrain raphe area have been shown to occurL In addition, due to proximity to the ejection site, the report of the local uptake of HRP into neuronal soma and dendrites 33 is also a consideration. The autoradiographic and H R P data at least suggest that the interconnection of contralateral homologous reticular nuclei permits an integration between the two sides of the reticular formation. Since adjacent reticular nuclei were also labeled, one may speculate that a connection between a nucleus receiving descending afferent projections (BR) and nuclei receiving spinal cord afterents (nGC) would permit integration of neural information at the brain stem level. Although following HRP injections into the nRPC, Sakai et al. 53 found a low density of HRP-positive neurons in the locus coeruleus (LC) and subcoeruleus area, we found only occasional HRP-labeled neurons in the LC area following ejections into any of the brain stem nuclei. Since Sakai et al. ~3 also observed similar labeling when the injections were made into the parabrachialis lateralis, it is possible that diffusion up the needle tract could account for the discrepancy. In the same study, these investigators 53 observed a median density of HRP-positive neurons in the dorsal posterior hypothalamic area. In our study, except in the case where an oblique approach was used to position the micropipette, no HRP-labeled cells were found in this region. Since Sakai et al. ~3 used a 45 ° approach, labeling of some other structure along the injection pathway may account for this difference. In our study, some afferent projections were found to be common to all three brain stem nuclei. For example, HRP-positive cells in the dorsal and ventral tegmenturn were found following ejections into the BR, nGC or nRPC. This pattern is consistent with the autoradiographic 11 and H R P 53 studies which have shown projections from the dorsal and/or ventral tegmentum to reticular nuclei. In addition, an electrophysiological study 1° has demonstrated a direct excitatory influence of the dorsal tegmentum on the nGC. We observed HRP-labeled cells in the deep layers of the superior colliculus following ejections into the reticular nuclei, which is consistent with many anatomical a,22,5a and physiological46,47,5s studies demonstrating monosynaptic tectoreticular connections. However, HRP-positive cells were also routinely observed in deep tectal layers following ejections into the BR. Since this study appears to be the first to report such a connection, the possibility of a projection from this area must await confirmation using other (degeneration, autoradiographic) techniques. All three brain stem nuclei were also found to receive afferents from the medial vestibular nucleus. This is in accord with degeneration 27,56, HRPSa and electrophysiological45,46 studies which give evidence for afferent pathways from the medial as well as various combinations of the other three (superior, lateral and descending) vestibular nuclei. In agreement with previous studies s,27, we saw no reactive cells in the cerebellar cortex. However, two recent H R P studies 2,53 in addition to our own, have failed to find HRP-positive cells in the fastigial nucleus of the cerebellum despite well-documented anatomical s,62 and physiologicaP 4,19 evidence for such a projection. It is possible that fastigio-reticular cells represent a special subpopulation of neurons which are unable to visibly take up and/or transport H R P as has been suggested for

273 some cells in the caudoputamen 41. Finally, in agreement with studies of the DR using HRP 2 and autoradiographic techniques 4a, we have failed to observe any transport to the caudate nucleus from the BR area, a connection which has been suggested by Usunoff et al. 59 using degeneration techniques. When BR afferent projections, as determined in the present study, are compared with BR efferent projections 5,v very different patterns of distribution are found. While efferent projections from the BR descend predominantly to the spinal cordS, 7,1~, afferent projections to this nuclear group seem to be derived largely from structures rostral to the pons, notably the mesencephalic central gray matter and surrounding tegmentum. These afferents from the central gray lend neuroanatomical support to the recent suggestion 5 that the direct actions of opiates in the mesencephalic central gray 2°,44 as well as electrical stimulation of this structureaS,4a may produce analgesia, at least in part, by activating a descending inhibitory system that is relayed to the primary afferents through the raphe magnusS, 49. In contrast, considerable overlap appears to exist between afferent and efferent projections of the nGC. In addition to providing reciprocal interconnections between the reticular nuclei 61 (and present study), the nGC both sends and receives spinal cord fibers. This suggests that the nGC may serve as a relay connection in a feedback loop between the spinal cord and the brain stem reticular formation. Since information can also be fed into the nGC from the adjacent nuclei, some mechanism for integration appears possible at this brain stem level. However, as observed previously 51 and in the present study, the nRPC lacks these spinal afferent connections, and may serve other functions.

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