Afferent Projections to the Rat Locus Coeruleus as Determined by a Retrograde Tracing Technique JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN Yale University School of Medicine and Connecticut Mental Health Center, 34 Parkstreet, New Haven, Connecticut 06508

ABSTRACT

Afferent connections to the rat locus coeruleus (LC), which contains exclusively noradrenergic neurons, have been traced using the technique of retrograde transport of horseradish peroxidase (HRP). In order to ensure accurate placement of adequate amounts of HRP in the LC, a microiontophoretic delivery technique coupled with single cell recording was employed. The use of electrophysiological "landmarks" as aids in placing the injections is described. Following HRP injections into the LC, forebrain structures containing labelled neurons included the insular cortex, the central nucleus of the amygdala, the medial, lateral and magnocellular preoptic areas, the bed nucleus of the stria terminalis, and the dorsomedial, paraventricular and lateral hypothalamic areas. In the brainstem reactive neurons were observed in the central grey substance, the reticular formation, the raphe, vestibular, solitary tract and lateral reticular nuclei. In particular, the areas of catecholamine cell groups A,, A2 and A5 appeared to contain many reactive cells. Labelled neurons were also observed in the fastigial nuclei and in the marginal zones of the dorsal horns of the spinal cord. This pattern of afferent innervation supports suggestions for a role for the LC in behavioral arousal mechanisms and autonomic regulation.

In the rat, the locus coeruleus (LC) appears to consist entirely of norepinephrine-containing neurons (Dahlstrom and Fuxe, '65; Kuhar e t al., '72; Koslow e t al., '72; Swanson and Hartman, '75; Swanson, '76a). In fact, nearly half the NE-containing neurons of this species appear to be located in the LC (Swanson, '76a). The LC is the source of an extensively ramifying plexus of fine axons innervating the entire cerebral (Ungerstedt, '71; Maeda and Shimizu, '72; Kobayashi et al., '74; Tohyama e t al., '74; Pickel e t al., '74; Lindvall and Bjorklund, '74; Jones and Moore, '77) and cerebellar (Ungerstedt, '71; Olson and Fuxe, '71; Pickel e t al., '74) cortices and the hippocampus (Ungerstedt, '71; Pickel et al., '74; Jones and Moore, '77). The LC also projects to parts of the amygdala (Ungerstedt, '71; Pickel e t al., '74; Jones and Moore, '771, septa1 nuclei (Ungerstedt, '71; Pickel e t al., '741, hypothalamus (Ungerstedt, '71; Maeda and Shimizu, '72; Kobayshi et al., '74; Jones and Moore, '77) thalamus (Maeda and Shimizu, '72; Lindvall e t al., '74; Jones and Moore, '77), lower J. COMP. NEUR. (1978) 178: 1-16.

brainstem (Loizou, '69; Pickel e t al., '74) and the spinal cord (Kuypers and Maisky, '75; Hancock and Fougerousse, '76; Nygren and Olson, '77). In fact, axon collaterals of a single LC neuron may reach several widely separated areas of the brain (Ungerstedt, '71; Tohyama e t al., '74; Nakamura and Iwama, '75). Although the anatomy of the efferent projections of the LC is fairly well understood, until recently a comprehensive picture has not been available concerning the afferent projections to the LC. In an early degeneration study, Russel ('55) suggested that the LC receives input from: the underlying reticular formation and the adjacent central grey, collaterals of fibers descending in the dorsal longitudinal fasciculus of Schutz, the principal sensory and mesencephalic nuclei of nV, collaterals from the commissure of the lateral lemniscus, and an unidentified, but postulated input from the amygdala. More recently afferents, to the LC have been demonstrated from the fastigial nucleus (Snider, '75) reticular

2

JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN

formation (Scheibel and Scheibel, '73; Shimizu and Imamoto, '70) and the serotonergic neurons of the raphe nuclei (Conrad e t al., '74; Bobillier et al., '76); as well as several forebrain and hypothalamic areas (Conrad and Pfaff, '76a,b; Saper e t al., '76; Swanson, '76b, '77; Swanson and Cowan, '77). I t has also been speculated that functional interconnections exist between cells of the LC and other brainstem catecholaminergic nuclei. Although early fluorescent-histochemical studies detected the presence within the LC of catecholamine-containing terminal varicosities (Dahlstrom and Fuxe, '65; Fuxe, '65), and more recently Lindvall and Bjorklund ('74), using the glyoxylic acid technique, have demonstrated axon collaterals from the ascending central tegmental catecholamine bundle directed towards the LC, the precise origin of these terminals has not been determined. I t is also possible that these terminals represent collaterals of LC neurons themselves (Swanson, '76a; Shimizu and Imamoto, '70). Hokfelt and co-workers ('74), using an immunohistochemical technique, have recently identified epinephrine-containing neurons in the area of the lateral reticular nucleus and the solitary tract nucleus which seem to project to the LC. Finally, physiological evidence has been obtained that sensory stimuli are somehow relayed to the LC. Noxious stimuli applied anywhere on the body transiently accelerate the firing of LC neurons, even in the anesthetized animal (Korf et al., '74). This activation is followed by a short quiescent interval before the cell resumes its previous rate of firing (Cedarbaum and Aghajanian, '76). Thus there is evidence that, in addition to having widespread projections, the LC receives input from diverse areas of the nervous system. I t was therefore decided to undertake a systematic study of the afferent projections of the LC in the albino rat, using the technique of retrograde transport of horseradish peroxidase (HRP) (Kristensson et al., '71: LaVail and LaVail, '72). A similar study on the cat LC was recently reported by Sakai and associates ('76), while this manuscript was in preparation. However, the NE-containing neurons of the cat LC are scattered in the pons and midbrain throughout the central grey matter, the dorsolateral tegmentum and the area surrounding the brachium conjunctivum (Chu and Bloom, '74; Jones et al., '74) and are intermingled with non-catecholamine-con-

taining cells. On the other hand, since the rat LC appears to consist of a clustered, homogeneous population of NE-containing neurons (Dahlstrom and Fuxe, '65; Swanson and Hartman, '75; Swanson, '76a) afferent projections to the LC in this species are more likely to relate specifically t o the NE containing cells. Furthermore, in the study of Sakai et al. ('76) no projections to the LC from the spinal cord or from areas rostra1 to the hypothalamus were mentioned. Because of the small size of the rat LC we used a microiontophoretic technique for the delivery of HRP (Graybiel and Devor, '74; Bunney and Aghajanian, '76; Aghajanian and Wang, '77) to achieve extremely dense deposits and to minimize spread to other structures. We have also combined this delivery technique with single-cell recording to facilitate accurate localization and placement of the HRP injection. METHODS

Male Sprague-Dawley rats (Charles River Labs) weighing 260-300 g were used. For placement of the HRP, the rats were anesthetized with chloral hydrate (400 mg/kg, i.p.1 and mounted in a stereotaxic instrument equipped with non-traumatic ear bars. A 3mm burr hole was drilled in the skull over the LC, centered 1.1mm posterior and 1.1 mm to the right of the midline. The dura was carefully removed, and a two-barreled micropipette assembly for single unit recording and iontophoresis of HRP was lowered into the brain using a hydraulic microdrive. Because the LC of the rat is a very small structure (approximately 1.0 mm in rostrocaudal extent, and only 0.4 mm in width and 0.6 mm in depth where it attains its greatest cross-sectional diameter near its caudal pole), we found that the use of single-cell recording greatly aided in guiding the placement of the HRP deposit. The dual microelectrode assembly consisted of a fine-tipped (1 pm tip diameter) micropipette electrode for unit recording and a coarser (40-60 pm tip diameter) electrode for iontophoresis of HRP. The iontophoresis barrel was bent a t the shoulder to a 30' angle and affixed alongside the recording electrode with dental acrylic cement so that their distal shafts were parallel and the recording electrode extended 1 mm ahead of the HRP pipette. This arrangement allowed the recording electrode to be used to explore the vicinity of the LC t o determine optimum

3

LOCUS COERULEUS AFFERENTS

placement for the HRP injection with minimum local tissue damage from the much larger HRP containing barrel, thus minimizing the potential for uptake of HRP by damaged axons merely passing through the area. Both pipette barrels were'loaded with a few strands of fiberglass prior to pulling to allow rapid filling of the tips by capillary action (Tasaki e t al., '68). The recording electrode was filled with 2 M NaC1. The other barrel contained a 25% solution of HRP (Sigma Grade VI) in 0.01 M NaC1. Impedances were typically 3-5 M R in the recording barrel and 5-20 MR in the HRP barrel. For some control experiments in which exact placement of the HRP deposit was not critical, the HRP was ejected from a single micropipette electrode. The signal from the recording electrode was amplified a n d displayed by conventional methods. The HRP solution was connected to a high voltage constant current source, which provided either a continuous or a pulsed positive current. The prolonged ejection periods used tended to cause the impedance of the electrode to rise markedly with time, presumably due to polarization of the electrode tip (Graybiel and Devor, '74). Use of the pulsed current source (7 seconds on-time alternating with 7 seconds off-time) was found to allow longer ejections with minimum current blocking. As the electrode descended through the cerebellum dorsal to the LC, layers of intense electrical activity corresponding to the granule and Purkinje cell layers were traversed. This succession was broken, a t a depth of approximately 5 mm below the skull surface, by a zone of electrical silence corresponding to the location of the fourth ventricle. If the electrode was directed into the LC the numerous biphasic (positive-negative) action potentials typical of LC cell firing began to appear a t a level of 5.5-5.8 mm below the skull surface. As described previously the pattern of firing was fairly regular, with a rate of 0.5-5 Hz (Graham and Aghajanian, '71; Cedarbaum and Aghajanian, '76). The frequency of cell firing could be transiently increased by noxious stimulation such as pressure applied with a hemostat to the contralateral hind paw. When the electrode penetration was made laterally or slightly anterior to the LC, the large cells of the mesencephalic nucleus of nV were encountered. A few of these cells were spontaneously active, but the majority were quiescent. However, all could be activated by

gently depressing the mandible. Once the site for HRP deposition was determined, the micropipette assembly was advanced so that the tip of the HRP-containing pipette came to lie within the LC. The HRP was then ejected, using a positive current of 4 pA, either for 10 to 20 minutes a t two sites within the LC approximately 200 p m apart or a t a single site approximately in the center of the nucleus for 30 minutes. HRP injections were placed in the right LC of six animals. Four animals were injected in the caudal pole of the nucleus, where the majority of its constituent neurons are located (Swanson, '76a) and two injections were made into its rostra1 end, where several afferent inputs have been reported to terminate (Conrad and Pfaff, '76a,b; Swanson and Saper, '75; Swanson, '76a,b; Saper e t al., '76). Control injections were placed in the cerebellum dorsal to the LC (n = 31, and in the reticular formation ventral, and ventromedial to the nucleus (n = 2) and in the medial parabrachial area (n = 1).One animal served as a n uninjected control. Twenty-two to twenty-six hours post-injection, the animals were reanesthetized and perfused through the heart with 900 cc of a 0.05 M phosphate buffer (pH 7.3) containing 1%paraformaldehyde and 1% glutaraldehyde. The brain and spinal cord were carefully removed and stored overnight a t 4°C in 0.05 M Tris buffer (pH 7.61, containing 5% sucrose. The next morning 50-pm frozen frontal sections of the brain and representative areas of the cervical, thoracic and lumbar portions of the spinal cord were cut and processed by a modification (Aghajanian and Wang, '77) of the method of Graham and Karnovsky ('66). The sections were examined under darkfield illumination (Kuypers e t al., '74). This mode of illumination allowed for rapid yet accurate scanning of the sections for reactive cells, which were identified by their content of coarse, highly refractile granules, and were easily distinguishable from both vascular endothelial cells (Nauta e t al., '71) as well as non-vascular elements possessing endogenous peroxidase activity (Keefer and Christ, '76). RESULTS

LC injections In the six LC-injected animals the HRP deposit was confined to the area of the pons known to contain noradrenergic neurons (fig. 1).The center of the injection site in the LC was rendered opaque by the dense HRP reac-

4

JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN

Fig. 1 (left) Fluorescence micrograph of rat locus coeruleus. The LC appears as a compact cluster of brightly fluorescing cells lateral to the fourth ventricle (V4)and medial to the mesencephalic nucleus of the trigeminal nerve (Vm). (right) Darkfield photomicrograph of HRP injection site in the locus coeruleus. Note that the blackened zone, which corresponds to the area of dense HRP deposit, blankets the entire area containing fluorescent cells in the left hand picture. The deposit is so dense that it has lost its refractility, thus appearing black even in darkfield. It is set off by a "halo" of much less dense accumulation of HRP in surrounding areas. Cafibration bar, 200 g m

tion product (fig. I). This opaque zone was usually 400-800 pm in transverse diameter, and tended to conform to the elongated contours of the LC, probably due to limitation of diffusion by surrounding fiber tracts. Axons of LC neurons emanating from the injection site were observed to contain anterogradely transported HRP (Lynch et al., '73). In the midbrain these fibers coalesced into a compact bundle corresponding to the dorsal NE bundle (Ungerstedt, '71). In some animals fibers could be traced rostrally as far as the septa1 nuclei and caudally beyond the decussation of the pyramids. Fibers could be observed to cross the midline a t the level of the anterior LC, in the decussation of the brachium conjuctivum, and in the posterior commissure (Lindvall and Bjorklund, '74; Jones and Moore, '77). In all six animals with LC ejections HRP reactive neurons were identified in many brain areas as well as in the spinal cord as summarized in table 1. As indicated, cells were found on both sides of the midline; however some areas did not contain labelled cells in all animals studied. Figure 3 represents a series of frontal sections showing the distribu-

tion of HRP reactive neurons in the brain c typical animal, the injection of which is ill trated in figure 1. The description which j lows will emphasize areas from the forebr, to the spinal cord in which neurons w located in all animals. Four telencephalic areas were consisten seen to project to the LC. In all LC-injec animals, reactive pyramidal cells were served in layer 5 of the insular cortex (an 13 and 14: Krieg, '46) dorsal to the rhi sulcus, ipsilateral to the side of the inject and extending from the level of approximat A7400 to A9000 in the coordinates of KO. and Klippel ('63: fig. 3a). This projection v very sparse, and usually no more than one two cells per section were seen. In three a mals with injections covering more anter areas of the LC scattered cells were obseri bilaterally in the prefrontal cortex as well Basal forebrain structures containing re tive neurons included the ipsilateral bed 1 cleus of the stria terminalis (fig. 3b), the m nocellular and lateral (fig. 3b) preoptic are Small multipolar reactive cells were seen some animals bilaterally in the medial a

5

LOCUS COERULEUS AFFERENTS TABLE 1

Distribution of LC afferents Ipsilateral

Ipsilateral

> contralateral

=

Ipsilateral contralateral

Contralateral > ipsilateral

Contralateral only

I. Telencephalon

11.

111.

IV.

V.

VI.

Prefrontal cortex Insular cortex Bed nucleus stria terminals Amygdala, central nucleus Medial pfeoptic area Lateral preoptic area Magnocellular preoptic area Periventricular preoptic area Diencephalon Hypothalamus: Dorsomedial nucleus Paraventricular nucleus Ventromedial nucleus Lateral hypothalamus Perifornical area Central grey Parafascicular nucleus Mesencephalon Dorsal raphe nucleus Central grey Lateral lemniscus Reticular formation Pons Locus coeruleus Raphe nuclei Reticular formation Parabrachial nuclei Principal sensory nucleus nV Medulla Vestibular nuclei Nucleus reticularis lateralis Nucleus tractus solitarius Nucleus parasolitarius Nucleus commissuralis Nucleus prepositus Spinal nucleus nV Reticular formation Raphe nuclei Cerebellum Nucleus fastigius

+ +-++ +++ +

++

(+)

++ ++ (+I + + +

+

+ t-+t

+ +

*

+

+ +

++-+++

++ ++

+

++

+ + +

++

Distribution of reactive neurons in rat brain following HRP injection into the locus coeruleus. The density of labelled cells is indicated as fOllows: + 0-1cells per section; 1.5 cells per section; + + + more than five cells per section. Parentheses indicate that area was not labelled in all animals. “Ipsilateral” and “contralateral” refer to location of reactive cells with respect to side of injected LC (*I.

++

periventricular preoptic area, albeit with a marked ipsilateral preponderance. All these areas were more heavily labelled in those animals with more anterior injections. Many reactive neurons were found in the central nucleus of the amygdala ipsilateral t o the injection in all animals (figs. 2, 3c,d). Most of these cells were located in the dorsal and medial portions of the posterior half of the nucleus. No reactive neurons were observed in any other amygdaloid nuclei, although an occasional labelled cell was observed in the substantia inominata medial to the central nucleus. Scattered reactive cells, either small (15-25

g ) and polygonal or slightly larger and fusi-

form or bipolar, extended posteriorly from the lateral preoptic area through the lateral hypothalamus (fig. 3d). Other hypothalamic areas containing labelled neurons included the perifornical area, the paraventricular nucleus, (magnocellular portion) (fig. 3c), the dorsomedial (3 animals) and the ventromedial (2 animals) nuclei. In the latter nucleus cells were only observed ipsilateral to the injection; in the dorsomedial and paraventricular nuclei a few cells were distributed contralaterally as well. In the midbrain the majority of HRP-reac-

6

JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN

Fig. 2 Darkfield photomicrograph of HRP-reactive neurons in central nucleus of the amygdala. Calibration bar, 150 fim.

tive neurons were located within the central gray substance (figs. 3e-g), predominantly ipsilateral to the side of HRP injection. These predominantly small, multipolar cells tended to occur for the most part around both tectal and tegmental segments of the dorsal longitudinal fasciculus of Schutz, along the lateral border of the central gray, and immediately beneath the ependymal lining of the aqueduct of Sylvius. Larger, round-to-oval reactive cells were seen within the dorsal raphe nucleus. Occasional reactive cells were also observed along the lateral lemnisci of both sides (figs.

3f,g). In three animals reactive neurons were observed in the median raphe nucleus. Relatively few cells were found labelled in the pons in any of the animals with LC injections and only one or two cells per animal were observed in the contralateral LC (fig. 3h). At the pontomedullary junction, cells were observed lying dorsally and laterally to the superior olive, medial to the exiting fibers of the facial nerve (figs. 3h-j). Reactive neurons were also seen bilaterally in the n. pontis oralis, immediately adjacent to and on both sides of the facial nerve (fig. 3j). Labelled neurons were seen in all vestibular nuclei, the n. prepositus hypoglossi and the fastigial nucleus of the cerebellum, all with a predominantly contralateral distribution (figs. 3j,k). Scattered cells were observed as well in the medullary reticular formation (n. reticularis gigantocellularis), in the medullary raphe nuclei and in the lateral portions of the spinal nucleus of the trigeminal nerve of both sides (figs. 3k-n). As shown in figures 31-11 several portions of the solitary tract complex contained labelled cells. These cells, which occurred with equal frequency on both sides of the brain, were easily distinguishable from the many, presumably glial elements in the solitary and commissural nuclei possessing endogenous DAB reactivity (Keefer and Christ, '76), in which the reaction product appeared finely granular and brown in color as opposed to the coarse, brilliant HRP granules. Several different cell types were observed in the various nuclei: Large round-tofusiform reactive neurons were observed in the lateral solitary tract nucleus, (parasolitarius) beginning near its rostra1 pole. These cells were large, round-to-oval in shape and were bipolar or multipolar. Farther posteriorly, smaller, multipolar reactive cells appeared within the medial solitary nucleus LC a t just

Abbreviations A, aqueduct of sylvius ace, central n. of amygdala ap, area postrema CA, anterior commissure CAI, internal capsule CC, corpus callosum cgs, central grey substance cn, commissural nucleus F, fornix f, fastigial n. FLD, dorsal long. fasciculus FMT, mammilothalamic tract

FOR, reticular formation FR, fasciculus retroflexus HI, hippocampus hl, lateral hypothalamus IC, inferior colliculus io, inferior olivary n. lc, locus coeruleus LL, lateral lemniscus MFB, medial forebrain bundle ML, medial lemniscus nps, n. parasolitarius

nts, n. tractus solitarius nVII, cranial n. VII OT, optic tract pf, parafascicular n. ph, n. prepositus pol, lateral preoptic n. pom, medial preoptic n. poma, magnocellular preoptic n. pvh, paraventricular hypothalamus RS, rhinal sulcus rl, lateral reticular n. vmh, ventromedial hypothalamus

LOCUS COERULEUS AFFERENTS

7

E

8

JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN

as well as in the white matter along the lateral edge of the nuclear areas of the dorsal horns (fig. 5).

Control ejections The distribution of HRP reactive neurons in the brain following control ejections into several areas around the LC is presented in table 2. As can be seen, there is considerable overlap between the LC injections and the controls. Virtually all areas positive for the LC were labelled as well from a t least one control site. In two animals ejections remained strictly confined t o the cerebellum dorsal to the LC. In these animals the majority of reactive neurons were observed in the following areas: the parietal cortex ipsilateral to the injection, the contralateral pontine nuclei, the contralatera1 inferior vestibular nucleus, the medullary reticular formation (n. reticularis parvocellularis), the tail of the contralateral inferior olive, bilaterally in the lateral reticular nucleus. However, the cells labelled in the lateral reticular nucleus following cerebellar inFig. 4 Typical reactive neurons in the nucleus reticularis lateralis. a. Large, multipolar neurons seen after both LC and control injections into the cerebellum. b. Fusiform neurons seen after LC injections only. Calibration bar, 50 pm.

anterior to the level of the obex. This column of cells continued into the commissural vagal nucleus, and extended in some animals into the central grey matter dorsal to the central canal of the upper cervical segments. No reactive neurons were seen in the dorsal motor vagal nucleus. In all animals reactive neurons were seen in the n. reticularis lateralis ipsilateral to the injection site (figs. 31,m). These cells tended to be of three morphological types: large (30-40 Fm diameter), spherical and multipolar (fig. 4a) small (15-25 p diameter) and multipolar; and medium sized and spindle-shaped (fig. 4b). The latter conform to the descriptions of catecholamine-containing neurons in the area (Dahlstrom and Fuxe, '65; Hokfelt, '74). Ten representative sections were taken from the spinal cord a t each of three levels: the cervical and lumbar enlargements and the midthoracic area. Reactive neurons were observed in at least one such section per animal. These cells were observed for the most part contralateral to the injection in the Rexed's Lamine I (Steiner and Turner, '72;Waibl, '73)

Fig. 5 Darkfield photomicrograph of dorsolateral quadrant of cervical spinal cord. Two HRP-reactive neurons (arrows) are seen in the marginal zone of the dorsal horn (dh). Dashed line indicates approximate boundary between grey matter and the lateral funiculus (fl). Calibration bar, 200 pm.

9

LOCUS COERULEUS AFFERENTS TABLE 2

Distribution of reactive cells in rat forebrain following LC us. control injections Site LC

I. Telencephalon Prefrontal cortex Insular cortex Parietal cortex Bed'nucleus stria terminals Amygdala, central nucleus Amygdala, medial and basal n.uclei Preoptic area: Medial Periventricular Magnocellular Lateral 11. Diencephalon Hypothalamus Dorsomedial nucleus Ventromedial nucleus Paraventricular nucleus Lateral hypothalamus Perifornical area Central grey Parafascicular nucleus 111. Mesencephalon Superior colliculus Inferior colliculus Substantia nigra Central grey Dorsal raphe nucleus Reticular formation IV. Pons Locus coeruleus Raphe nuclei Reticular formation Parabrachial nuclei Principal sensory nucleus nV Pontine nuclei V. Medulla Vestibular nuclei Cochlear nuclei Nucleus reticularis lateralis Nucleus tractus solitarius Nucleus parasolitarius Nucleus commissuralis Nucleus prepositus Inferior olive Spinal nucleus nV Reticular formation Raphe nuclei Area postrema VI. Cerebellum Fastigial nucleus Dentate nucleus

(+I

++ +-

Reticular formation

+ ++

(+I

Medial parabrachial nucleus

+ + + + + +

+ + + +

+ ++ +

+ + + + + +

++ ++-

-

+ + + + +

-

+

-

(+)

-

-

+ + + + + + + +-

++ **

-

-

+

+-

-

+ +

++-

++ +

+ + +

-

-

+ + +- * + + + + + +

+ +

Comparison of distribution of HRP-reactive neurons in rat brain following LC and control ejections. belled cells; - absence of labelled cells: ( + I cells present In some animals, but not in others. * Large and small multipolar cells only. ** Reactive neurons included a population of mediumsized, fusiform cells.

jections were never of the spindle-shaped variety. In one animal t h e HRP injection was placed in the vicinity of the medial parabrachial nu-

Cerebellum

+ + + + + -

+ ++

+ + + ++*

+ + +-

+ + + + + + indicates presence of la

cleus, lateral to but not extending into the mesencephalic nucleus of nV. The distribution of reactive neurons throughout the brain of this animal was remarkably similar to t h a t

10

JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN

found following LC injections. However, in addition numerous reactive cells were observed in the ipsilateral dentate nucleus of the cerebellum and all parts (zona compacta, zone reticulata and pars lateralis) of the ipsilateral substantia nigra. In a fourth animal the HRP was injected into the cerebellum and spread along the brachium conjunctivum. The distribution of reactive neurons in the brain of this animal'was similar to that following injection into the parabrachial area. Following injection of HRP into the reticular formation ventral to the LC in two animals reactive neurons were again identified in several areas in common with the LC. However, no reactive cells were identified in the dorsal raphe nucleus or in the medial nucleus of the solitary tract. Only one cell in one animal was found in the central nucleus of the amygdala. Areas containing reactive neurons not found following injections into the LC included the contralateral cochlear nuclei, the inferior colliculi and the auditory cortex (area 41: Krieg, '46). Many cells were also observed throughout the prefrontal cortex bilaterally and many reactive neurons in the spinal cord were found in the ventral horns as well as in the marginal zones of the dorsal horns. DISCUSSION

By the use of a combined electrophysiological and microiontophoretic technique we have achieved highly localized, extremely concentrated ejections of HRP into the locus coeruleus of the albino r a t for the purpose of retrograde tracing of its afferent connections. The use of a fine tipped glass microelectrode delivery system of maximum tip diameter of 40-60 p m further minimized the possiblity of damage to local structures, thereby increasing the specificity of the injections t o terminal structures within and immediately surrounding the LC. However, in interpreting the results of studies utilizing HRP as a tracer, one must always consider the possibility that some retrogradely labelled cells may have accumulated tracer protein via damaged or even undamaged axons passing through or near the injection site. Following these injections, HRP-containing neurons were observed in widespread areas of the brain. However, many of these projections were relatively sparse; some areas contained less than one reactive neuron per section, and some did not contain reactive cells in all

brains studied. For purposes of discussion, these afferent areas can be grouped into three categories: (1) forebrain afferent areas; (2) brainstem nuclei; and (3) spinal cord.

Forebrain afferent areas The one area of the neocortex which was consistently found to project to the LC was a highly restricted band occurring around and dorsal to the rhinal sulcus, in areas 13 and 14 (Krieg, '46). This group of cells appeared to comprise a portion of a larger field projecting to the surrounding reticular formation. Such a projection to the LC has not previously been reported, either by classical degeneration methods or in the HRP study by Sakai et al. ('76) of LC afferents in the cat. The central nucleus of the amygdala was found to contain many reactive cells in all animals. Although fibers have been traced from the amygdala into the medial forebrain bundle using classical techniques (Cowan et al., '65) neither the precise origins nor the terminations of this pathway were shown. Recently, Hopkins ('76) has shown that the central nucleus projects widely to lateral areas of the reticular formation throughout the length of the brainstem, and a projection to the substantia nigra has been revealed as well (Bunney and Aghajanian, '76). In agreement with Hopkin's observation that more lateral areas of the reticular formation receive heavier projections from the central nucleus than do more medial areas, more reactive cells were found in the central nucleus when HRP was injected lateral to the LC, and none were seen when the injection was placed in the ventromedial reticular formation. The central nucleus of the amygdala receives a very dense noradrenergic innervation from the LC (Pickel e t al., '74; Jones and Moore, '77). However, the significance of this reciprocal relationship between these areas and the LC remains unknown. We also observed afferents to the LC from several basal forebrain areas, including the bed nucleus of the stria terminalis, and the medial and lateral preoptic areas. Projections to the LC from these areas have also been traced autoradiographically by other workers following the anterograde transport of tritiated amino acids injected into these nuclei (Swanson and Saper, '75; Swanson, '76b; Conrad and Pfaff, '76a). The presence of reactive cells in the dorsomedial (Swanson and Saper,

LOCUS COERULEUS AFFERENTS

'75) and paraventricular (Conrad and Pfaff, '76b; Swanson, '77) nuclei of the hypothalamus is also consistent with the results of autoradiographic and immunohistochemical studies. Projections from the paraventricular nucleus may represent collaterals of fibers originating in the paraventricular nucleus and continuing to autonomic centers in the lower brainstem and spinal cord (Swanson, '77; Saper e t al., '76; Hancock, '76; Kuypers and Maisky, '75). We observed few HRP positive neurons in the ventromedial nucleus of the hypothalamus (Conrad and Pfaff, '76b; Saper et al., '76) and none in the anterior hypothalamus (Conrad and Pfaff, '76a) which have also been reported to project to the LC. No reactive neurons were seen in our material in the arcuate, suprachiasmatic or subthalamic nuclei as reported by Sakai e t al. ('76) in the cat. However, as these authors noted, the different distributions of hypothalamic afferents to the LC in the rat and cat may be a function of species differences.

Brainstem afferents We found HRP-reactive neurons in the dorsal raphe nucleus, as well as occasional cells scattered along the midline raphe of the pons and medulla. Cells of the raphe nuclei have been shown to contain serotonin (Dahlstrom and Fuxe, '65). Serotonin-accumulating nerve terminals (Leger and Descarries, '76) and terminals containing tryptophan hydroxylase (Pickel e t al., '76, '77) are present in the LC, and there is biochemical evidence suggesting that the serotonin-containing neurons of the midbrain raphe exert an inhibitory influence on the LC (Kostowski e t al., '74; Lewis et al., '76). Recent autoradiographic studies have also provided evidence for projections from the raphe nuclei to the LC (Conrad et al., '74; Bobillier e t al., '76). However, these findings must be interpreted with caution in light of the proximity of the LC to the supra-ependymal plexus of serotonergic nerve endings lining the fourth ventricle (Lorenz and Richards, '75). We failed to observe any direct afferents to the LC from the substantia nigra. Sakai e t al. ('76) reported that reactive neurons were observed in the brains of their cats when HRP was injected into the "subcoeruleus" area or into the so-called "LCa" area between the subcoeruleus and the "principle LC," but not when the HRP was injected into the "princi-

11

ple LC" alone. We did, however, observe labelled cells in all parts of the substantia nigra following injections into the medial parabrachial area or into the reticular formation ventral to the LC. However, in the rat these areas contain few if any noradrenergic neurons (Dahlstrom and Fuxe, '65; Swanson and Hartman, '75) so, a t least in this species, the existence of a connection between the substantia nigra and the noradrenergic neurons of the LC is doubtful. In all animals, reactive cells were observed, dorsal and lateral to the superior olive, and along the exiting course of the facial nerve. Interestingly, the A5 catecholamine cell group of Dahlstrom and Fuxe ('65) is located in this area. Medullary areas which contained HRPpositive cells following injection in the LC included the nucleus reticularis lateralis and the nucleus of the solitary tract. Catecholamine cell groups, designated A1 and A2, respectively in the nomenclature of Dahlstrom and Fuxe ('65) are located in these areas. Recently Hokfelt and coworkers have demonstrated that a t least a portion of these neurons contain the enzyme phenylethano1amine-Nmethyl-transferase (PNMT); hence they may be capable of synthesizing epinephrine and utilizing it as their transmitter. Axons of this PNMT-containing system have been traced to the ventral portion of the LC (Hokfelt et al., '74). High concentrations of epinephrine (Koslow and Schlumpf, '74) as well as of PNMT (Saavedra e t al., '74) are present in the areas of groups A1 and A2 as well as in the LC, and we have recently demonstrated an a-adrenoreceptor-mediated inhibition of LC cell firing by epinephrine (Cedarbaum and Aghajanian, '76). Of course, no conclusions can be drawn regarding the aminergic nature of efferents to the LC using the HRP method alone. Studies combining the use of HRP with histochemical procedures for the demonstration of monoamines would be most helpful in this regard, as i t is possible that non-catecholamine neurons in these areas project to the LC. Two areas of the solitary tract nuclei contained reactive cells. The medial nucleus, which is continuous with the commissural nucleus of the vagus nerve, is known to receive inputs from cortical and aortic baroreceptors (cf., Reis et al., '75; Calaresu, '75; for reviews). The area lateral to the solitary tract itself (n. parasolitarius) has also been shown to receive gustatory afferents (Makous e t al., '63). Also

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JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN

of note are the projections to the LC from the vestibular and fastigial nuclei. The LC projects widely to the cerebellar cortex (Olsen and Fuxe, '71; Ungerstedt, '71; Pickel e t al., '74). I t is therefore possible that some of these connections might represent those of a LC-cerebellum-LC feedback loop.

In this study we have confirmed the existence of afferent projections to the LC and vicinity described by several previous workers, as well as identifying several heretofore unknown inputs. However, it must be noted that no areas were identified which project to the LC exclusive of all surrounding areas, except perhaps the fusiform possibly catecholSpinal cord aminergic cells of the lateral reticular nuReactive neurons were observed in the cleus, although surrounding regions appear to marginal zones of the dorsal horns of the receive inputs from areas not projecting to the spinal cord. Large neurons in these areas are LC. It is unlikely that this extensive overlap sensitive to noxious stimuli (Christensen and results from lack of specificity of the techPerl, '70; Kumazawa et al., '73) and give rise nique, as our injections were highly localized to slowly-conducting fibers which do not ter- and minimal local tissue damage was prominate in the specific sensory areas of the duced. It would appear instead t h a t the LC is thalamus, but rather seem to end in areas "wired in parallel" with several neuronal syswhose functions are associated with the nox- tems relaying both sensory information to ious rather than discriminative properties of forebrain areas as well as descending mespainful sensation (Kerr, '75). Likewise, noci- sages from the forebrain to lower centers. The noradrenergic neurons of the LC appear ceptive information may reach the LC from the spinal nuclei of the trigeminal nerve. De- capable of influencing the activity of vast generating terminals, probably representing areas of the brain via their widely ramifying collaterals of spinoreticular fibers, have been axonal projections. The LC has been impliobserved in the subcoeruleus area following cated as playing a role in a variety of funcanterolateral cordotomy in monkeys (Mehler tions and behaviors, ranging from micturation e t al., '60). Such connections may represent (Kuru and Yamamoto, '64) respiration (Johnthe anatomical substrate underlying the re- son and Russel, '52) control of cerebral blood flow (Raichle et al., '75) and cardiovascular sponse of LC neurons to noxious stimuli. The LC thus appears to be under the influ- regulation (Ward and Gunn, '76a,b; Przuntek ence of a variety of types of input from: (1) and Philippu, '73) to sleep and waking (Jouvet forebrain structures such as the neocortex, and Delforme, '65; Jones e t al., '73; Lidbrink, amygdala and hypothalamus; (2) other brain- '74) and learning and memory (Zornetzer and stem monoaminergic neurons, including sero- Gold, '76). Information converging on the LC tonergic neurons of the midline raphe nuclei may be integrated and relayed over its widely and catecholamine (norepinephrine or epi- diverging projections throughout the brain, nephrine) containing neurons in the pons and signalling t h a t a change in state of the organmedulla; (3) a variety of sensory relay areas. ism has occurred. Such a role fits well within The first group, especially the various hypo- the conceptual framework of the central thalamic centers, is of interest with regard to noradrenergic system as a mediator and intepotential interactions involving the LC and grator of behavioral arousal and autonomic functions such a s regulation of the autonomic function (Bolme et al., '72). nervous system. Connections in the second ACKNOWLEDGMENTS category may represent in part the anatomiWe wish to express our gratitude to Ms. cal substrate for the interactions of drugs acting on the various monoamine systems. Fi- Annette Lorette, Ms. Nancy Margiotta and nally, the existence of sensory afferents to the Ms. Faye Gomes for their excellent technical LC may provide an added insight into the assistance. This work was supported in part by function of the LC. In addition to receiving Grants MH-17871, MH-14459 and the State of input from the spinal cord, the LC may receive Connecticut. gustatory information as well as information LITERATURE CITED about the status of the cardiovascular system F. 1971 Esterotaxis troncoencefalicade la Abad-Alegria, from the solitary tract nuclei. Auditory inforrata. Trab. Inst. Cajal Invest. Biol., 63: 193-224. mation may come from the scattered cells Aghajanian, G . K.,and R. Y. Wang 1977 Habenular and along the lateral lemnisci. other midbrain raphe afferents demonstrated by a

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Afferent projections to the rat locus coeruleus as determined by a retrograde tracing technique.

Afferent Projections to the Rat Locus Coeruleus as Determined by a Retrograde Tracing Technique JESSE M. CEDARBAUM AND GEORGE K. AGHAJANIAN Yale Unive...
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