A’euroscience

Vol. 41, No. Printed in Great Britain

2/3, pp. 52S542,

0306-4522/91$3.00+ 0.00 Pergamon Press plc 0 1991IBRO

1991

ADRENERGIC INNERVATION OF THE RAT NUCLEUS LOCUS COERULEUS ARISES PREDOMINANTLY FROM THE Cl ADRENERGIC CELL GROUP IN THE ROSTRAL MEDULLA V. A. PIERIBONE*~ and G. ASTON-JONES$§ SDivision of Behavioral Neurobiology, Department of Mental Health Sciences, Hahnemann University, Broad and Vine, Philadelphia, PA 19102-l 192, U.S.A. *Department of Biology, New York University, Washington Square, New York, NY 10003, U.S.A. Abstract-Focal iontophoretic injections of the retrograde tracer Fluoro-Gold into the locus coeruleus were combined with immunocytochemistry for phenylethanolamine N-methyltransferase, the final enzyme in the synthesis of epinephrine. Retrograde labeling confirmed recent findings that the major afferents to the locus coeruleus are present in the ventrolateral (nucleus paragigantocellularis) and dorsomedial medulla (nucleus prepositus hypoglossi), areas containing the Cl and C3 adrenergic cell groups, respectively. The Fluoro-Gold label revealed morphologic details of locus coeruleus afferent cells. Labeled neurons in the prepositus hypoglossi region were typically round (10 pm diameter) or ellipsoidal and compressed against the ventricle wall (10 x 20 pm), while those in the paragigantocellularis were most often multipolar and ellipsoidal or triangular in shape (10 x 2&20 x 30 pm). Double labeling in the same tissue sections revealed that locus coeruleus afferent neurons are intercalated among phenylethanolamine N-methyhransferase-positive Cl and C3 neurons. Twenty-one per cent of locus coeruleus afferent neurons in paragigantocellularis stained for phenylethanolamine N-methyltransferase while only 4% of locus coeruleus afferents in the prepositus hypoglossi area exhibited phenylethanolamine N-methyltransferase immunoreactivity. In paragigantocellularis, doubly labeled neurons were usually the smaller locus coeruleus afferents, while in the prepositus hypoglossi phenylethanolamine N-methyltransferase labeling was found in all cell types that project to the locus coeruleus. Phenylethanolamine N-methyltransferasepositive fibers from the Cl and C3 cell groups form an adrenergic fiber bundle in the dorsomedial medulla; in the pons, these fibers appear to exit this bundle and innervate the locus coeruleus. Fibers from the neurons of the C3 cell group also appear to ascend on the dorsal surface of the medulla to innervate the locus coeruleus. The phenylethanolamine N-methyltransferase fiber innervation in the locus coeruleus was dense and highly varicose. Phenylethanolamine N-methyltransferase innervation in the dorsal pons was not restricted to the locus coeruleus but was also prominent in neighboring areas such as Barrington’s nucleus and the lateral dorsal tegmental nucleus of Gudden.

Several lines of evidence indicate that there is a prominent adrenergic innervation of the locus coeruleus (LC). The LC is densely populated with alpha, adrenergic receptors, and iontophoretic adrenaline acting at these sites potently inhibits spontaneous activity of LC neurons.*‘9 In addition, Hokfelt et ~1.~’ reported phenylethanolamine Nmethyltransferase-immunoreactive (PNMT-IR) fiber

TPresent address: Department of Histology and Neurobiology, Karolinska Institutet, Stockholm S-104 01, Sweden. §To whom correspondence should he addressed. Abbreviations: DBH, dopamine p-hydroxylase; FG, FluoroGold; -IR, -immunoreactive; LC, locus coeruleus; LDT, lateral dorsal tegmental nucleus of Gudden; PHA-L, Phaseolus vulgaris-leucoagglutinin; PGi, nucleus paragigantocellularis; PNMT, phenylethanolamine Nmethyltransferase; PrH, nucleus prepositus hypoglossi; RITC, rhodamine isothiocyanate; Sol, nucleus tractus solitarius; TBS, Tris-buffered saline; TBSS, TBS containing 1% normal goat serum; WGA-HRP, wheatgerm agglutininconjugated with horseradish peroxidase. 525

labeling in LC and others have found that monoamine@ terminals form synapses with LC neurons.‘8.‘9 More recently, PNMT-IR synapses onto LC somata and dendrites have been reported.30 Immunocytochemical staining revealed a PNMTIR fiber tract that arises from the three medullary adrenergic cellgroups (ClC3) and follows a dorsomedial direction as it projects rostrally.23~24 Electrolytic lesions of this ascending adrenergic fiber bundle eliminated the majority of PNMT-IR fibers in LC4 indicating that they originate in the medulla. Anatomic’ and physiologic’2-‘4 studies indicate that major afferents to LC arise from the nucleus paragigantocellularis (PGi), which contains the Cl adrenergic cell group, and from the nucleus prepositus hypoglossi (PrH), which contains C3 adrenergic neurons. No projections from the nucleus tractus solitarius (Solw2 cell group to LC were found, indicating that the adrenergic innervation of the LC could derive from either Cl, C3 or both. However, the precise origin of the adrenergic innervation of LC has remained unclear; previous studies combining

V. PI. PIERIBONL and Cr. ASTON-JONFS

526

retrograde transport with immunocytochemistry to examine catecholamine inputs to LC have been conflicting (see Discussion) and the results hard to interpret. To further characterize adrenergic innervation of the LC area, we have utilized an improved double labeling method employing iontophoretically deposited Fluoro-Gold (FG)‘4.42 combined with immunofluorescence for PNMT. EXPERIMENTAL

Fluoro-Gold

PROCEDURES

injections

A detailed description of the use of FG, and its combination with immunofluorescence, is found elsewhere.‘4 Briefly, male Sprague-Dawley rats (n = 31: Hilltop Labs and Taconic Farms) weighing 300-350 g were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic instrument. An incision was made in the scalp, a small hole drilled in the skull and the dura retracted. The LC was approached caudally from a 17 -angle to avoid rupturing the overlying tranverse sinus. Glass capillary tubes (1.5 mm o.d., Omega Dot, Glass Co. of America) were cleaned with distilled water and ethanol and made into micropipettes with tips of IO pm diameter. Just prior to use, pipettes were filled with 1% FG in 0.1 M sodium acetate buffer (pH 3.3). Recordings of single- and multi-cellular activity were obtained from the injection pipette. Once LC was tentatively identified by its characteristic discharge the electrode was connected to a constant properties,’ current source and FG was iontophoretically deposited. Current (f I .O PA) was pulsed (4 s on, 4 s off) for 2--10 min. The electrode remained in place 5 min before and after the injection, and the animal was perfused five days later. Others?’ have found that PNMT staining may be augmented by colchicine pretreatment. Therefore, in two animals 150 pg of colchicine in 10 ~1 of saline was injected into the lateral ventricle 48 h prior to perfusion. Animals were perfused with 80ml of saline at a rate of lOOml/min. This was followed by 500ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at the same rate, and an additional 500 ml at 30 ml/mitt; perfusion solutions were at room temperature. Brains were removed and immersed in fixative for 90 min at 4 C. Prior to sectioning on a freezing microtome, brains were blocked and immersed in 20% sucrose in 0.1 M phosphate buffer at 4 ‘C overnight, while brains sectioned on a Vibratome (Technical Products International) were cut immediately after the 90-min postfixation. Forty-micrometer-thick, serial, coronal sections of the medulla were collected in cold isotonic phosphate-buffered saline (pH 7.4). Sectioning and subsequent immunohistochemical processing of tissue sections were performed under low ambient light to retard fading of the FG. Every section was collected from the area postrema to the VIIth nerve exit, and alternate sections were collected through the LC. Following two rinses in Tris-buffered saline (TBS: 0.1 M. pH 7.6). sections to be examined exclusively

Cl C2

c3 DR IO IML LDt LC mlf

for FG labeling were mounted on actd cleaned, gelatinized slides and allowed to air-dry overnight. These sections were coverslipped in DPX (BDH, Poole. U.K.). All coverslipped slides containing fluorescent compounds were stored in the dark at --20°C.

Sections containing FG that were also processed for immunofluorescence were treated as follows. After sectioning. free-floating sections were rinsed twice in TBS and incubated for 30 min in TBS containing 0.25% Triton X- 100 and 3% normal goat serum. Polyclonal antibodies raised in rabbit against either bovine (Eugene Tech.) or rat adrenal PNMT were tested; results were better with the latter which are reported here. The antisera directed against rat adrenal PNMT was generously provided by Dr Martha Bohn; its production and specificity has been tested and reported elsewhere.’ Antisera were diluted 1: 1000 in TBS containing I”& normal goat serum (TBSS) and 0.5% Triton X-100. Sections containing LC were incubated in PNMT antisera or in a solution of rabbit anti-dopamine-beta-hydroxylase (Eugene Tech., NJ. U.S.A.) diluted I:500 in TBSS. For control experiments, sections containing FG retrograde labeling were incubated in normal rabbit serum (1:200). Incubations were for 4 6 h at room temperature in humidified chambers with gentle agitation. Following two rinses in TBSS, tissue was processed by one of two methods. (1) Sections processed for immunofluorescence were incubated for 45 min in a solution of lissamide-rhodamine isothiocyanate (RITC)-conjugated, affinity purified, goat anti-rabbit antisera (Boehringer Mannheim, Indianapolis, IN, U.S.A.) in TBSS (1:40) and rinsed twice in TBS. (2) Sections processed for light-stable staining were subjected to peroxidase-antiperoxidase; diaminobenzidine immunocytochemistry. For the latter. after incubation in primary antisera, sections were incubated in a dilute solution of swine anti-rabbit antisera (I :40; Dako. CA, U.S.A.) for 1h, rinsed in TBS, and transferred to a solution of rabbit peroxidase-antiperoxidase (1: 100: Dako) for 2 h. After a short rinse in TBS, sections were transferred to a solution of diaminobenzidine and HzO, (0.04 and 0.15%, respectively) in phosphate buffer (pH 6.0) for 2-5 min. Some sections were subjected to goldsubstituted silver intensification of diaminobenzidine. as previously described.” Following mounting from TBS and overnight drying all tissue was dehydrated through a graded series of ethanols, cleared with xylenes and coverslipped in either DPX

(Gallard Schlcenger; fluorescent material) or Permount (Fisher; light-stable material). Sections were examined and photographed using a Nikon Microphot microscope. FG was visualized with U.V.illumination (330-nm excitation peak) and RITC with green illumination (530-nm peak). Black-and-white photographs of fluorescent material were made using Kodak Plus-X film (ASA 125) while light-stable material was photographed using Kodak Panatomic X film (ASA 32). The microscope was connected by a video link to a Nikon/Joyce-Lobe1 Magiscan for plotting cells and anatomUsing a motorized stage under computer ical boundaries

adrenergic cell group in ventrolateral medulla adrenergic cell group in Sol and dorsal motor nucleus of the vagus adrenergic cell group in the area of PrH dorsal raphe nucleus inferior olivary complex intermediolateral cell column of the spinal cord lateral dorsal tegmental nucleus of Gudden locus coeruleus medial longitudinal fasciculus

PGi PrH PY RVL Sol SP5 VII nVI1 XII

nucleus paragigantocellularis nucleus prepositus hypoglossi pyramidal tract rostra1 ventrolateral medulla solitary tract nucleus spinal trigeminal nucleus facial nucleus facial nerve hypoglossal nerve nucleus

Adrenergic LC afferents control and specialized software developed in the laboratory of Dr M. T. Shipley, several adjacent microscope fields of a single tissue section could be drawn using a x 10 objective and then combined to produce a single drawing of a microscope field as though seen under a x 2 objective. Additionally, injection site volume was estimated using densitometry functions of the computer. By using alternate illumination wavelengths, cells containing FG, RITC immunofluorescence, or FG plus immunofluorescence could be discriminated and plotted. LabeIed neurons were counted in sections (approximately 23 sections) taken from the caudal third of the facial nucleus to approximately 1 mm caudal to the facial nucleus, at the rostra1 aspect of the lateral reticular nucleus. Locations and nomenclature of anatomical structures were taken from the atlas of Paxinos and Watson.3’ RESULTS Retrograde labeling from

Eocus coeruleus injections

All injections were centered in LC as determined either by immunofluorescence in the same sections for dopamine /I-hydroxylase (DBH) or by examination of the same sections under dark-field illumination (which readily reveals the LC nucleus). injections lasting 5 min appeared to optimally fill the LC nucleus (Fig. 1); shorter injection times produced deposits that did not fill the entire nucleus and longer

521

injection times produced injections that encroached on surrounding nuclei, especially parabrachial, Barrington’s, lateral dorsal tegmental of Gudden (LDT) and vestibular nuclei. Optimal injections yielded an estimated injection volume of 0.13 mm3 (equivalent to a cube 500 pm on each side). Animals with injection sites that apparently spread beyond LC proper were not analysed as they may give rise to erroneous retrograde labeling in PGi and elsewhere (see Discussion). There was no necrosis at injection sites, and an iontophoretic deposit of FG in LC did not decrease the intensity or number of DBH-stained neurons in LC (Fig. 1). The pattern of retrograde labeling obtained here was equivalent to that seen in our previous studies using FG3’ or wheat-gem a~lutinin-conjugated horseradish peroxidase (WGA-HRP5). Retrogradely labeled cells were predominantly located in two areas, both in the rostra1 medulla: the perifascicular region of PrH located dorsomedially, and the PGi situated ventrolaterally; these afferents are described in greater detail immediately below. Small numbers of cells were seen in the dorsal cap of the paraventricular nucleus of the hypothalamus and adjacent to the spinal central canal.

Fig. I. A representative iontophoretic injection of FG in LC. (A) Ultraviolet epi-illuminated photomicrograph showing the dense core of the FG deposit in LC. (B) Photomicrograph of the same section as in A illuminated to show RITC immunofluorescence for DBH. Note that the injection site is restricted to the cellular bounds of the LC nucleus and that the presence of the injected FG does not result in necrosis or loss of DBH immunoreactivity. Medial is to the left and dorsal is at the top. Scale bar = 100 pm.

Fig, 2. Retrograde labeling following injection of FG into LC. A frontal section through the contralateral (A) and the ipsilateral (B) PrH in the dorsal medial medulla illuminated to reveal FG-containing neurons. Asterisk indicates the IVth ventricle. Scale bar = 50 pm. (C) A frontal section through the rostra1 medulla illuminated to reveal neurons in ipsilateral PGi retrogradely labeled with FG from an injection in the LC. Medial is to the left and dorsal is at the top. Arrow indicates ventral surface of the brain. Scale bar = IOOIcm.

530

V. A. RERIBONE and G. ASTON-JONES

Fig. 4. Representative cell types retrogradely labeled in PrH following injections of FG into LC. (A) Scattered LC afferent neurons in the interstitial nucleus of the medial longitudinal fasciculus subjacent to the PrH. These cells are similar to those in the PrH proper but with processes oriented dorsoventrally. (B-D) Examples of the predominant cell type retrogradely labeled in the PrH ventral to the IVth ventricle. Such cells are small (about 15 pm in diameter) and ellipsoidal to round with processes oriented mediolaterally. Scale bar = 10 pm.

Afferents to the locus coeruleus from the dorsomedial medulla FG-labeled cells in the dorsomedial medulla appeared caudally at the rostra1 border of XII and extended rostrally to the germ of the VIIth nerve (see Ref. 33, frontal plates 6763). Labeled neurons were most concentrated immediately under the IVth ventricle in caudal, medial, suprafascicular PrH, but did not extend into the lateral or supragenial PrH. Scattered labeled neurons extended ventrally to lie along the dorsolateral border of the medial longitudinal fasciculus (mlf) (Fig. 2A, B). Retrograde labeling was bilateral with a slight contralateral dominance. The predominant morphologies for cells retrogradely labeled in the dorsomedial medulla (PrH area) are represented in Figs 3-5. Neurons were typically ellipsoidal (approximately 10 x 20 pm), often appearing compressed against the ventricle wall, or round (approximately 10 pm in diameter). Overall, LC-projecting neurons in the PrH area were similar to one another in size and shape; there were no large or triangular neurons in this region retrogradely labeled from LC. Afferents to the locus coeruleus in the ventrolateral medulla Retrograde labeling in the ventrolateral medulla was restricted to the cytoarchitectonic boundaries of PGi as described by Andrezik et al2 (Fig. 2C). Labeled cells appeared caudally at the transition between the lateral reticular nucleus and PGi, and extended rostrally to the area media1 to the caudal

Fig. 5. Representative cell types retrogradely labeled in PGi following injections of FG into LC. (A-C) Medum to small, oval, multipolar neurons were the most common LC afferents in PGi, along with small ellipsoidal neurons. (D, E) Larger, multipolar neurons were seen in a column of cells lateral to the main body of retrogradely labeled neurons. (F) Ellipsoidal neurons with few processes (< 4) were found in the PGi as well as in the medial longitudinal fasciculus subjacent to PrH. Scale bar = 10 pm.

third of the facial nucleus (Ref. 33, plates 6964). Medially, labeled cells extended to the border of the inferior olivary complex and laterally to the border of the spinal trigeminal nucleus; dorsally, labeled cells were bounded by the nucleus ambiguus while ventrally they extended to the surface of the brain. Labeling was strongly ipsilateral with only occasional cells in the contralateral PGi. The predominant morphologies for LC-projecting neurons in PGi are indicated in Figs 3 and 5. Most commonly, labeled cells were multipolar and often ellipsoidal or triangular in shape (approximately 10 x 20 pm). Many labeled neurons, especially those located laterally, contained large triangular or ellipsoidal somata (approximately 20 x 30 pm; Fig. 5D, E). Thus, in contrast to LC afferents in the dorsomedial medulla (described above), a variety of morphologically different cells project to LC from the ventrolateral medulla. Immunocytochemical staining As shown in Figs 6-8, PNMT staining patterns in the medulla were similar to those previously The Cl adrenergic cells in the reported. 2325.28,36.39

Fig. 6. A bright-field photomicrograph of a frontal hemisection of the rostra1 medulla processed with peroxidase-antiperoxidase immunocytochemistry to show PNMT immunoreactivity. Three distinctive adrenergic cell groups are evident: the Cl cell group in the ventral medulla, the C2 cell group in the dorsolateral medulla (Sol, dorsal motor nucleus of the vagus), and the C3 group of the dorsomedial medulla (PrH). Dorsal is at the top and lateral is to the right, Scale bar = 500 pm.

Adrenergk LC ail’erents

531

532

V. A. P~ERIB~NEand G. ASTON-JONES

Fig. 7. Bright-tield photomontage of PNMT-IR somata and processes in a frontal section of the dorsal medulla (peroxidase-antiperoxidase immunocytochexnistry). Two distinct groups can he identified in the dorsal medulfa: a medial group (C3) within the PrH and medial lon~tud~nal fasciculus (single arrows) and a lateral group (C2) within the Sol and dorsal motor nucleus of the vagus (double arrows). Scale bar = iO0pm. ventrolateral medulla were predominantly dispersed throughout PGi (as defined by Andrezik et al.‘) including the rostra1 aspect medial to the facial nucleus (Figs 6, 8; however, see Refs 24-28). In addition, Cl neuronsz3 extended caudally into the lateral reticular nucleus. Incubation in the primary antibody was limited to 4 h to minimize possible wash-out of FG from retrogradely labeled neurons. Although background imm~ofluores~nce was increased shghtly as a result, there was no apparent reduction in PNMT or DBH immunoreactivity (Fig. 1). Also, processing for immunofluorescence did not alter the number of FGlabeled neurons. Injections of colchicine into the lateral ventricle of two animals increased PNMT immuno~activity only slightly, and such increased staining appeared to be limited to the Cl area (see also Ref. 39). A caudal cell column and rostra1 cell cluster were identified in Cl with or without colchicine treatment, similar to results of others following colchicine.” PNMT-IR neurons in the dorsal medulla appeared to form a medial group within the medial longitudi-

nal fasciculus and suprafascicular PrH area (C3), and a separate lateral group in the Sol and the dorsal motor nucleus of the vagus (CZ), as previously described.“s2’ Fibers from the Cl and C2 cell groups entered a tract that ascends through the dorsomedial medulla (Fig. 8). Cl fibers entered this tract at the level of the rostra1 three-quarters of the Cl cell group (Fig. 8A-C). This tract projected through the medulia and into the pons where, at the level of rostra1 LC, fibers coursed dorsafly to innervate the entire LC nucleus (Fig. 9A, B) and the adjacent pericoerulear region including Barrington’s nucleus-LDT. PNMTIR fibers within this adrenergic longitudinal bundle were aggregated into smaI1 discrete groups, so that the bundle was composed of numerous fascicles (Fig. 1DB). PNMT fiber staining in the medulla and pons revealed two rostrally projecting fiber paths from C3 cells. (1) C3 fibers traveled rostrally along the ventral surface of the fourth ventricle above the medial longitudinal fasciculus. At the Sevel of LC, fibers extended laterally, above the dorsal tegmental

Adrenergic LC aRerents

Fig. 8. Computer-assisted plot of PNMT-IR structures in the medulla and pons. Six representative frontal hemisections ordered sequentially from the caudal medulla (A) to the rostra1 pons (F). Note that fibers from both the Cl and C3 cell groups (neurons indicated as closed squares) send processes into the adrenergic longitudinal bundle (B, C) and that at the level of rostra1 LC (F) fibers from this bundle exit dorsally to enter and ramify throughout the LC nucleus as well as the central gray. Also note that fibers from the C3 cell group send rostromedially projecting fibers that travel ventrally to the fourth ventricle.

and through the LDT to innervate LC (Fig. 8D, F). (2) Fibers from cells of the C3 group were also seen entering the longitudinal adrenergic bundle at the level of the C3 cell bodies (Fig. 8C). nucleus

Andrenergic locus coeruleus afferent neurons in the dorsomedial medulla

FG-positive, LC afferent neurons were closely intermingled with PNMT-IR C3 cells in the dorsomedial medulla, predominantly in the region of the suprafascicular caudal PrH (Fig. 11). However, LC afferents and adrenergic neurons were not equally distributed in this region: C3 cells extended farther caudally, dorsal to the XIIth nerve nucleus where they became continuous with medial cells of the C2 cell group. In contrast to the relatively high percentage of LC afferents in PGi that were also PNMT-IR (see below), only a few LC-projecting neurons in PrH stained for PNMT. Counts revealed that approximately 4% of the FG-labeled neurons (11 of 301 bilaterally) in the dorsomedial medulla also exhibited PNMT immunoreactivity; conversely, 3% of PNMT-IR neurons (11 of 376) in the C3 area contained FG (Figs 11, 13). Neurons doubly labeled for both PNMT and FG (Fig. 11) included all cell types indicated in Figs 3 and 4.

Adrenergic locus coeruleus aflerent neurons in the ventral lateral medulla

Neurons retrogradely labeled with FG were closely intermingled with PNMT-IR neurons of the Cl cell group, throughout its middle and rostra1 aspects (Fig. 14). However, the Cl cell cluster extended farther caudally (into the level of LRt) than did retrogradely labeled cells, which were restricted to PGi. We found that many putatively adrenergic neurons of the Cl cell group project to the ipsilateral LC. In addition, areas of the Cl cell group differed in the fraction of LC afferent neurons that contained the adrenergic marker. In mid-Cl about 18% of FGlabeled neurons exhibited PNMT immunoreactivity (Figs 12, 14). In the very rostra1 Cl area (medial to the caudal third of the facial nucleus) there were fewer LC-projecting neurons present, but a much higher percentage of them were doubly labeled (80%). Overall, cell counts revealed that 21% (81 of 378) of the neurons in the ipsilateral PGi area that contained retrogradely transported FG also exhibited PNMT immunoreactivity. Conversely, 10% (81 of 779) of the PNMT-IR neurons in the same PGi area also contained FG. Occasionally neurons were also doubly labeled in contralateral Cl.

B Fig. 9. PNMT-IR innervation of LC. (A) Dark-field photomicrograph of a frontal section through mid-K showing PNMT-IR fibers. Note the extensive fiber labeling restricted to the LC. Scale bar = 100 pm. (B) High-power bright-field photomicrograph of PNMT-IR in LC. Note the numerous immunoreactive varicosities and terminals. Asterisks in both A and B denote the IVth ventricle. Scale bar = 50pm. 534

Adrenergic LC afferents The predominant cell type that was both retrogradely labeled and PNMT-IR in PGi was small and ellipsoidal (approximately 20 pm in the long axis) with only a few processes (see Figs 5A, 12). The PNMT-IR neurons that project to LC formed a rather homogeneous subpopulation of LC afferents in PGi; no large or triangular LC afferents contained PNMT. In addition, in the Cl area in particular FG-labeled PNMT-IR neurons contained only light or moderate levels of FG fluorescence, while neurons exhibiting intense FG labeling never contained PNMT immunoreactivity (Fig. 12C, D).

distinct populations of PNMT-IR neurons rostra1 dorsal medulla, as described above.

535 in the

Adrenergic pathways to locus coeruleus

The present results for PNMT-IR fiber staining indicate a pathway for adrenergic projections from the ventral lateral medulla to LC. According to our proposed pathway, Cl axons enter the adrenergic longitudinal bundle at the level of Cl somata, and course rostrally within this bundle. At the level of rostra1 LC, Cl fibers appear to exit this bundle and ramify throughout the adjacent LC nucleus. This proposed pathway is consistent with studies showing that lesions of the ascending adrenergic bundle eliminate the majority of PNMT immunoreactivity in DISCUSSION LC.“ The remaining PNMT-IR fibers in LC following the lesions could originate from the few PNMT-IR We report here that adrenergic neurons of the Cl neurons in the C3 or contralateral Cl cell groups that and C3 cell groups within PGi and PrH, respectively, project to LC. A minority of LC afferent neurons in innervate LC. The pathway described here for adrenergic innerPGi (21 X) and PrH (4%) appeared to be. adrenergic while the majority of afferents from each area were vation of the LC from the ventrolateral medulla was not disclosed in recent studies of projections from the not PNMT-IR. No direct projections to LC were seen ventrolateral medulla to LC using the anterograde from Sol (or C2 neurons) or the dorsal motor nucleus tracer PHA-L.2’.“r Instead, other pathways, not seen of the vagus. with PNMT staining here and presumably conveying Medullary afferents to locus coeruleus other signals to LC, were revealed by robust PHA-L This report confirms and extends earlier work5,35 fiber labeling. The present results, in light of those previous findings, indicate that multiple pathways identifying major afferents to LC from the ventroexist for fiber projections from the PGi to LC. lateral (PGi) and dorsomedial (PrH) medulla. While Together these findings also indicate a limitation of a previous study’ used WGA-HRP, the present the PHA-L technique, i.e. that either some cells do study achieved similar results using a non-lectin retrograde tracer. The pathway from PGi to LC has not take up and transport this tracer (e.g. adrenergic also been confirmed using anterograde transport of Cl neurons) or that the sample size of labeled WGA-HRP’ and Phaseolus vulgaris-leucoagglutinin neurons is so small that even prominent projections, (PHA-L) from PGi2r such as the PNMT pathway disclosed herein may be The present study provides the first detailed undetected. description of the location and morphology of adrenergic and non-adrenergic LC afferent neurons in Effects of adrenergic input on focus coeruleus activity Recent work from this laboratory revealed that the medulla. These findings indicate that a variety of different neuronal cell types project to the LC electrical stimulation of PGi potently excites over from the PGi, possibly indicating that various 70% of LC neurons, and that such excitation could types of information are transmitted to LC from this be blocked by ventricular or local application of area. excitatory amino acid antagonists.‘2,‘4 Additional pharmacological studies revealed a possible adrenerAdrenergic cell groups gic influence from PGi as well: when PGi-evoked Using PNMT immunocytochemistry we have excitation of LC was blocked by excitatory amino identified three groups of PNMT-IR somata in the acid antagonists, more than 90% of LC neurons medulla, corresponding to the Cl, C2 and C3 cell became inhibited by electrical stimulation of PGi.14 groups described by others.2*25,27,28,36,39,46 We found This underlying inhibitory response could be specifithat the Cl cell group is contained predominantly cally antagonized by intravenous or local application within the PGi described by Andrezik et aL2 (howof the alpha, antagonist idazoxan (Astier, Ennis and ever, see also Refs 27, 28). Our findings agree with Aston-Jones, unpublished observations and Ref. 3a). those of previous investigators24.25 who used the term As adrenaline, a potent agonist at alpha, receptors, C3 to designate the group of PNMT-IR neurons of inhibits LC neurons,8,9 PGi-evoked inhibition of LC the PrH, interstitial nucleus of the medial longitudimay be mediated in part by C 1 adrenergic neurons in nal fasciculus, and the suprafascicular region of the PGi found here to project to LC. medulla, but not including the PNMT-IR neurons in In contrast to the effects of PGi activation, Sol and the dorsal motor nucleus of the vagus, the stimulation of the PrH area inhibited 82% of LC latter constituting the C2 cell group25 (see Fig. 7). neurons.‘5.‘6 This inhibition was blocked by local This distinction corresponds to the two spatially application of the GABA antagonist bicuculline but

536

V. A. P~ER~BONE and iii. ASTOK-JOMS

Adrenergic LC afi’erents

537

Fig. 11.(A) A single neuron in the PrH complex, located on the lateral border of the medial lon~tudinal fasciculus, illuminated to reveal FG retrogradely transported from LC. (B) The same neuron illuminated to show RITC immunofluorescence for PNMT. Scale bar = 50pm. was not affected by the alpha, antagonist idazoxan.” This is consistent with the present findings that only a small percentage of LC afferents in the dorsomedial medulla appear to be adrenergic and that the LC receives a dense GABAergic innervation.6*43

Double labeling The observation that retrogradely labeled neurons in FGi that stain for PNMT immunorea~ti~ty were always small and exhibited light FG fluorescence may give some insight to the innervation pattern of LC afferents in PGi. It may be that the amount of FG (and therefore the amount of fluorescence) contained within a retrogradely labeled cell is determined by a combination of two factors: (i) the number of terminals within the injection site arising from the particular cell (degree of arborization) and (ii} the rate at which the neuron takes up FG. It seems possible, therefore, that PNMT-IR LC afferent neurons in PGi do not arborize greatly or possess small numbers of sites that take up FG. ~orn~~ri~ons with preuious results Guyenet and Young2’ and Haselton and Guyenet** combined injections of rhodamine-conjugated latex microspheres into the LC-pontine gray area with PNMT immunofluorescence to investigate adrenergic afferents to LC. In the first of these papers,*’ the authors reported that 70% of “LC afferents” in the ventrolateral medulla contained PNMT-IR. However, in the subsequent report2* only 36% of LC

afferents in Cl were found to be adrenergic. It seems likely that the discrepancy between these studies, and between their results and those presented here, is due in part to the tracer used (latex microspheres) and the placement of the injections (outside of LC) by those investigators. The injection sites reported in those studies were centered outside of the LC within an area (LDT-Barrington’s nuclei) that receives substantial adrenergic input (Fig. lOA)““’ and WGA-HRP’- or PHA-L-la~led~ fiber projections from the ventroiateral medulla. Therefore, retrograde labeling in the ventrolateral medulla from injections into these pericoerulear structures may reflect adrenergic inputs to areas nearby but outside of LC. As injections of latex microspheres typically produce local damage with consequent uptake and transport by passing fibers, the retrograde labeling in those studies may also reflect transport by nonterminal fibers of passage. Using injections of True Blue combined with DBH immunofluorescence, Sawchenko and Swanson4’ reported that LC afferents in the ventrolateral medulla arise primarily from Al noradrenergic neurons. This discrepancy with the present findings may also result from the tracer used. True Blue has been shown to be taken up by non-terminal fibers of passage.‘@ Therefore, if True Blue diffused into the region of the medullary lon~tudinal bundle containing ascending Al fibers and/or areas adjacent to LC that receive Al inputs (e.g. parabrachial nuclei3*” or LDT29), then many

Fig. IO. PNMT immunoreactivity in rostra1 LC. (A) Dark-field photomicro~aph of a frontal section showing PNMT-IR fibers in and around rostra1 LC. Arrows indicate the dorsoventral extent of LC at this level. Note fibers from the longitudinal adrenergic bundle (lower left) exiting the tract to innervate LC and the surrounding central gray. Asterisk indicates the IVth ventricle. Scale bar = 1OOpm. (B) High-power photomicrograph of PNMT-IR fibers in the longitudinal adrenergic bundle at the level of LC. Fibers are contained in discrete fascicles (arrows) within the bundle. Scale bar = 10 pm.

Fig. 12 53%

Adrenergic

Fig. 13. Computer-aided

plots showing

LC afferents

co-distribution

of PNMT-IR

neurons

and LC afferent

neurons

in individual sections of the dorsomedial medulla. Neurons that fluoresced for FG only (0) or PNMT only (a), and neurons that contained both FG and PNMT fluorescence (B). are plotted. Sections are ordered from caudal (A) to rostra1 (D). DBH/True Blue-positive neurons would be expected. This possible problem is largely circumvented by our use of FG as the retrograde tracer, as it is not appreciably taken up by passing fibers.34*42Ambiguity may also arise from the close contiguity between adrenergic (Cl) and noradrenergic neurons (Al) which are interdigitated throughout a large portion of the ventrolateral medulla.46 In view of the fact that both Cl and Al neurons stain with antibodies to DBH, it seems possible that some of the doubly

labeled neurons observed in their report were, in fact, adrenergic Cl neurons. As we have not compared PNMT and DBH labeling in the medulla, we cannot address the possibility of a noradrenergic input to LC from Al neurons. Possible ,functions of medullary adrenergic neurons Overall, little is known concerning functions of brain adrenergic neurons (for review see Ref. 24), but there is considerable evidence for a role of the Cl

Fig. 12. Fluorescent photomicrographs of adrenergic LC afferents in the ventrolateral medulla. (A) A single neuron retrogradely labeled with FG in the ventrolateral medulla. Note the granular nature of FG, helpful in identifying retrogradely labeled cells. (B) The same neuron as in A illuminated to show RITC immunoreactivity for PNMT. This is the typical neuronal cell type doubly labeled with FG and PNMT-IR. (C) Three neurons retrogradely labeled with FG from LC, consisting of a small lightly labeled neuron (arrow) and two heavily labeled larger neurons (below). (D) The same field as in C but illuminated to show PNMT immunofluorescence. The small lightly labeled neuron is seen to contain PNMT immunofluorescence, but the larger neurons are not PNMT-IR. (E) An FG-labeled neuron immediately lateral to the inferior olive in medial PGi. (F) The same neuron as in E illuminated to show PNMT immunofluorescence. This again is the typical cell type doubly labeled with both FG and PNMT-IR in PGi. (G) A neuron in rostra1 PGi medial to the VIIth nucleus illuminated to show retrogradely transported FG. (H) The same neuron as in G illuminated to show RITC immunoreactivity for PNMT. Scale bar = 10pm.

V. A.

PIERIBONE and G. ASTON-JONES

Fig. 14. Computer-aided plots showing co-distribution of PNMT-IR neurons and LC afferent neurons in individual sections of the ventrolateral medulla. Neurons that fluoresced for FG only (0) or PNMT only (A), and neurons that contained both FG and PNMT fluorescence (m), are plotted. Inserts: low-power

hemisections showing levels of plots in A and D. Sections are ordered from caudal (A) to rostra1 (D). cell group in autonomic control: (i) stimulation of the area containing Cl adrenergic neurons increases arterial blood pressure and heart rate, (ii) lesions in this area decrease arterial pressure to levels seen after cervical spinal cord transection,37,38 and (iii) neurons in the ventrolateral medulla (including Cl neurons) innervate the hypothalamus and the intermediolateral cell column in the thoracic spinal cord,3’,36,‘7,46both of which are intimately involved in regulation of the autonomic nervous system. This overall view is consistent with the finding that afferents to the PGijCl area include many structures linked to autonomic function.2”s47 Although recent studies indicate that baroreflexes require excitatory amino acid mechanisms in the spinal cord, 32 additional evidence reveals that impulse activity of spinally projecting putative Cl adrenergic neurons is strongly linked to phasic and tonic cardiovascular events.““,45 Taken together, therefore, the data suggest that Cl adrenergic input to the spinal cord acts in tandem with an excitatory amino acid pathway to modulate sympathetic outflow.

The function of C3 adrenergic neurons in the PrH is unknown. A subset of these cells has been reported to innervate the spinal cord,24a.49 but the functional significance of that projection has not been studied. The projection to LC demonstrated here is the only other known target of these neurons. The present studies, in view of previous results, reveal a marked parallel between descending projections from PGi to the intermediolateral cell column of the spinal cord and those ascending from PGi to LC. For example, recent evidence” I4 indicates that the LC receives an excitatory amino acid input from PGi and the present studies reveal an adrenergic input to LC from this same area; studies reviewed above indicate that the intermediolateral cell column of the spinal cord also receives an excitatory amino acid and adrenergic input from the PGi area relaying cardiovascular information to the spinal cord.2o Excitatory amino acid input from PGi may be critically involved in relaying certain sensory stimuli to LC neurons.‘“.‘2.‘4 Interestingly, physiologic studies have found that LC

Adrenergic LC afferents impulse activity is often correlated with peripheral autonomic activity,” so that stimuli which activate the LC also activate the autonomic system, perhaps via ascending and descending excitatory amino acid and adrenergic pathways. It seems reasonable to speculate, therefore, that PGi functions to help co-ordinate and disseminate sensory-autonomic information via global peripheral (sympathetic) and central (LC) nervous systems. As part of each of these pathways, the Cl adrenergic neurons are in

541

a strategic position for modulating peripheral and central circuits important in adaptive behavioral responses. Acknowledgements-We

thank Jesus Luna, Michael Shiplay and John Gayler for image analysis software, and Elisabeth Van Bockstaele and Genevieve Go for their expert assistance in the laboratory. Additionally, we are grateful to Martha Bohn and Holli Bernstein-Goral for the generous gift of PNMT antisera. This work was supported by PHS grant NS24698.

REFERENCES 1.Aghajanian G. K., Cedarbaum J. M. and Wang R. Y. (1977) Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons. Bruin Res. 136, 57&577. 2. Andrezik J. A., Chan-Palay V. and Palay S. L. (1981) The nucleus paragigantocellularis lateralis in the rat. Comformation and cytology. Anat. Embryo/. 161, 355-371. 2a. Andrezik J. A., Chan-Palay V. and Palay S. L. (1981) The nucleus paragigantocellularis lateralis in the rat. Demonstration of afferents by the retrograde transport of horseradish peroxidase. Anat. Embryol. 161, 373-390. 3. Aschoff A. and Holhinder H. (1982) Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). I. A parametric study in the central visual system of the albino rat. J. Neurosci. Mefh. 6, 179-197. 3a. Astier B. and Aston-Jones G. (1989) Electrophysiological evidence for medullary adrenergic inhibition of rat locus coeruleus. Sot. Neurosci. Abstr. 15, 1012. evidence for the 4. Astier B., Kitahama K., Denoroy L., Jouvet M. and Renaud B. (1987) Immunohistochemical adrenergic medullary longitudinal bundle as a major ascending pathway to the locus coeruleus. Neurosci. Lett. 74, 132:138. 5. Aston-Jones G., Ennis M., Pieribone V. A., Nickel1 W. T. and Shipley M. T. (1986) The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network. Science 234, 734737. 6. Berod A., Chat M., Paut L. and Tappaz M. (1984) Catecholaminergic and GABAergic anatomical relationship in the rat substantia nigra, locus coeruleus, and hypothalamic median eminence: immunocytochemical visualization of biosynthetic enzymes on serial semithin plastic-embedded sections. J. Histochem. Cytochern. 32, 1331-1338. 7. Bohn M. C., Dreyfus C. F., Friedman W. J. and Markey K. A. (1987) Glucocorticoid effects on phenylethanolamine N-methyltransferase (PNMT) in explants of embryonic rat medulla oblongata. Deul Brain Res. 37, 257-266. 8. Cedarbaum J. M. and Aghajanian G. K. (1976) Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and activation by the a,-antagonist piperoxane. Brain Res. 112, 413-419. 9. Cedarbaum J. M. and Aghajanian G. K. (1977) Catecholamine receptors on locus coeruleus neurons: pharmacological characterization. Eur. J. Pharmac. 44, 375-385. 10. Chiang C., Ennis M., Pieribone V. A. and Aston-Jones G. (1987) Effects of prefrontal cortex stimulation on locus coeruleus discharge. Sot. Neurosci. Abstr. 13, 912. 11. Elam M., Svensson T. H. and Thoren P. (1986) Locus coeruleus neurons and sympathetic nerves: activation by cutaneous sensory afferents. Brain Res. 366, 254261. 12. Ennis M. and Aston-Jones G. (1986) A potent excitatory input to the nucleus locus coeruleus from the ventrolateral medulla. Neurosci. Lett. 71, 299-305. 13. Ennis M. and Aston-Jones G. (1987) Two physiologically distinct populations of neurons in the ventrolateral medulla innervate the locus coeruleus. Bruin Res. 425, 275-282. 14. Ennis M. and Aston-Jones G. (1988) Activation of locus coeruleus from nucleus paragigantocellularis: a new excitatory amino acid pathway in brain. J. Neurosci. 8, 3644-3657. 15. Ennis M. and Aston-Jones G. (1989) GABA-mediated inhibition of locus coeruleus from the dorsomedial rostra1 medulla. J. Neurosci. 9, 2973-298 1. 16. Ennis M. and Aston-Jones G. (1989) Potent inhibitory input to locus coeruleus from the nucleus prepositus hypoglossi. Brain Res. Bull. 22. 793-803.

17. Gallyas F., Giircs T. and Merchenthaler I. (1982) High-grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J. Histochem. Cytochem. 30, 1833184. 18. Groves P. M. and Wilson C. J. (1980) Fine structure of rat locus coeruleus. J. camp. Neural. 193, 841-852. 19. Groves P. M. and Wilson C. J. (1980) Monoaminergic presynaptic axons and dendrites in rat locus coeruleus seen in reconstructions of serial sections. J. camp. Neural. 193, 853-862. 20 Guyenet P. G. and Les Brown D. (1986) Nucleus paragigantocellularis lateralis and lumbar sympathetic discharge in the rat. Am. J. Physiol. 250, R1981-R1094. 21 Guyenet P. G. and Young B. S. (1987) Projections of nucleus paragigantocellularis lateralis to locus coeruleus and other structures in rat. Bruin Res. 406, 171-184. 22 Haselton J. R. and Guyenet P. G. (1987) PNMT neurons and the Cl cell group with projections to both locus coeruleus and spinal cord. Sot. Neurosci. Abstr. 13, 809. 23. Hijkfelt T., Fuxe K., Goldstein M. and Johansson 0. (1974) Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain. Brain Res. 66, 2355251. 24. Hiikfelt T., Johansson 0. and Goldstein M. (1985) Central catecholamine neurons as revealed by immunohistochemistry with special reference to adrenaline neurons. In Handbook of Chemical Neuroanatomy. Volume 2: Classical Transmitters in the CNS (eds Bjiirklund A. and Hokfelt T.), pp. 157-276. Elsevier, New York. 24a. Hoktelt T., Poster G. A., Johansson O., Schitzberg M., Holets V., Ju G., Skagerberg G., Palkovits M., Skirboll L., Stolk J., UPrichard D. and Goldstein M. (1988) Central phenylethanolamine N-methyltransferase-immunoreactive neurons: distribution, projections, fine structure, ontogeny and coexisting peptides. In Epinephrine in the Central Nervous System (eds Stalk J. M., UPrichard D. C. and Fuxe K.), pp. 10-31. Oxford University Press, New York.

.

542

V. A.

PIERIBONE

and G. ASTON-JONES

25. Howe P. R. C., Costa M., Furness J. B. and Chalmers J. P. (1980) Simultaneous demonstration of phenlyethanolamine N-methyltrasferase immunofluorescent and catecholamine fluorescent nerve cell bodies in the rat medulla oblongata. Neuroscience 52, 222992338. 26. Kalia M. and Fuxe K. (1985) Rat medulla oblongata. 1. Cytoarchitectonic considerations. J. cotnp. Neurol. 233, 285-307. 27. Kalia M., Fuxe K. snd Goldstein M. (1985) Rat medulla oblongata. II. Dopaminergic, noradrenergic (Al and A2) and adrenergic neurons, nerve fibers, and presumptive terminal processes. J. camp. Neural. 233, 308-332. 28. Kalia M., Fuxe K. and Goldstein M. (1985) Rat medulla oblongata. III. Adrenergic (Cl and C2) neurons, nerve fibers and presumptive terminal processes. J. camp. Neurol. 233, 333-349. 29. McKellar S. and Loewv A. D. (1982) Efferent uroiections of the Al catecholamine cell IerouoI in the rat: an autoradiographic study. Brain Res: 241; 11-29. - _ 30. Milner T. A., Abate C., Reis D. J. and Pickel V. M. (1989) Ultrastructural localization of phenylethanolamine N-methyltransferase-like immunoreactivity in the rat locus coeruleus. Bruin Res. 478, 1 15. 30a. Milner T. A., Joh T. H. and Pickel V. M. (1986) Tyrosine hydroxylase in the rat parabrachial region: ultrastructural localization and extrinsic sources of immunoreactivity. J. Neurosci. 6, 258552603. 31. Milner T. A., Morrison S. F., Abate C. and Reis D. J. (1988) Phenylethanolamine N-methyltransferase-containing terminals synapse directly on sympathetic preganglionic neurons in the rat. Brain Res. 448, 205-222. 32. Morrison S. F., Milner T. A. and Reis D. J. (1988) Reticulospinal vasomotor neurons of the rat rostra1 ventrolateral medulla: relationship to sympathetic nerve activity and the Cl adrenergic cell group. J. Neurosci. 8, 12861301. 33. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotuxic Coordinates. Academic Press, Sydney. 34. Pieribone V. A. and Aston-Jones G. (1988) The iontophoretic application of Fluoro-Gold for the study of afferents to deep brain nuclei. Bruin Res. 475, 2599271. 35. Pieribone V. A., Aston-Jones G. and Bohn M. C. (1988) Adrenergic and non-adrenergic neurons of the Cl and C3 areas project to locus coeruleus: a fluorescent double labeling study. Neurosci. Left. 85, 2977303. 36. Ross C. A., Armstrong D. A., Ruggiero D. A., Pickel V. M., Joh T. H. and Reis D. J. (1981) Adrenaline neurons in the rostra1 ventrolateral medulla innervate thoracic spinal cord: a combined immunocytochemical and retrograde transport demonstration. Neurosci. Lett. 25, 257-262. 37. Ross C. A., Ruggiero D. A., Park D. H., Joh T. H., Sved A. F., Fernandez-Pardal J., Saavedra J. M. and Reis D. J. f 19841 Tonic vasomotor control bv the rostra1 ventrolateral medulla: effect of electrical or chemical stimulation of the area containing Cl adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J. Neurosci. 4, 474494. 38. Ross C. A., Ruggiero D. A. and Reis D. J. (1985) Projections from the nucleus tractus solitarii to the rostra1 ventrolateral medulla. J. camp. Neural. 242, 51 I-534. 39. Ruggiero D. A., Ross C. A., Anwar M., Park D. H., Joh T. H. and Reis D. J. (1985) Distribution of neurons containing phenylethanolamine N-methyltransferase in medulla and hypothalamus of rat. J. camp. Neural. 239, 127- 154. 40. Sawchenko P. E. and Swanson L. W. (1981) A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques, Brain Res. 210, 3 l-5 1. 41. Sawchenko P. E. and Swanson L. W. (1982) The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat. Brain Res. Ret’. 4, 275 325. 42. Schmued L. C. and Fallon J. H. (1986) Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties. Brain Res. 377, 1477154. 43. Shipley M. T., Pieribone V. A., Aston-Jones G. and Ennis M. (1988) GABAergic innervation of the rat locus coeruleus. Sot. Neurosci. Abstr. 14, 406. 44. Sun M., Hackett J. T. and Guyenet P. G. (1988) Sympathoexicitatory neurons of rostra1 ventrolateral medulla exhibit pacemaker properties in the presence of a glutamate-receptor antagonist. Brain Res. 438, 2340. 45. Sun M., Young B. S., Hackett J. T. and Guyenet P. G. (1988) Reticulospinal pacemaker neurons of the rat rostra1 ventrolateral medulla with putative sympathoexcitatory function: an intracellular study in vitro. Brain Res. 442, 229-239. of central adrenergic pathways: 1. 46. Tucker D. C.. Saper C. B., Ruggiero D. A. and Reis D. J. (1987) Organization Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord. J. camp. New-o/. 259, 591603. E. J., Pieribone V. A. and Aston-Jones G. (1989) Diverse afferents converge on the nucleus 47. Van Bockstaele paragigantocellularis in the rat ventrolateral medulla: retrograde and anterograde tracing studies. J. camp. Neurol. 290, 561 -584. G. and Shipley M. T. (1989) Multiple projection pathways 48. Van Bockstaele E. J., Astier B., Pieribone V. A., Aston-Jones from the ventrolateral medulla to locus coeruleus in rat. Sot. Neurosci. Abstr. 15, 1013. neurons in the rostra1 medulla oblongata 49. Wesselingh S. L., Li Y.-W, and Blessing W. W. (1989) PNMT-containing (Cl 1 C3 groups) are transneuronally labelled after injection of herpes simplex virus type I into the adrenal gland. Nrurosci. Left. 106, 99-104. (Accepted 19 Juty 1990)