Lower brainstem afferents to the cat posterior hypothalamus: A double-labeling study

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Brain Research Bulletin, Vol. 24, pp. 437-455. 0 Pergamon Press plc, 1990. Printed in the U.S.A.

Lower Brainstem Afferents to the Cat Posterior Hypothalamus: A Double-Labeling Study K. SAKAI, Y. YOSHIMOTO, P. H. LUPPI, P. FORT, M. EL MANSARI, D. SALVERT AND M. JOUVET

De’partement de Mdecine

Exptfrimentale, INSERM U 52, CNRS UA 1195, UniversitP Claude Bernard 8 Avenue Rockefeller, 69373, Lyon Cedex 08, France Received 6 November

1989

SAKAI, K., Y. YOSHIMOTO, P. H. LUPPI, P. FORT, M. EL MANSARl, D. SALVERT AND M. JOUVET. Lower bruinstem afferents to the cat posterior hypothalamus: A double-labeling study. BRAIN RES BULL 24(3) 4311155, 1990.-Using a double-immunostaining technique with cholera toxin (CT) as a retrograde tracer, the authors examined the cells of origin and the histochemical nature of lower brainstem afferents to the cat posterior hypothalamus. The posterior hypothalamus, in particular the lateral hypothalamic area, receives substantial afferent projections from: substantia nigra, peripeduncular nucleus, ventral tegmental

area, periaqueductal grey, mesencephalic reticular formation, peribrachial region including the locus coeruleus complex, rostral raphe nuclei and the rostral part of the nucleus raphe magnus. In addition, a moderate number of retrogradely labeled neurons was found in: Edinger-Westphal nucleus, nucleus reticularis pontis oralis, nucleus reticularis magnocellularis, caudal lateral bulbar reticular formation around the nucleus ambiguus and lateral reticular nucleus and the nucleus of the solitary tract. The posterior hypothalamus receives: 1) dopaminergic inputs from A8, A9 and A10 cell groups; 2) noradrenergic inputs from A6 and A7 pontine, as well as Al and A2 bulbar cell groups; 3) adrenergic inputs from C 1 cell group in the caudal medulla; 4) serotoninergic inputs from the rostra1raphe nuclei (B6, B7 and B8 cell groups); 5) cholinergic inputs from the peribrachial region of the dorsal pontine tegmentum as well as from the nucleus reticularis magnocellularis of the medulla; 6) peptidergic inputs such as methionin-enkephalin, substance P, corticotropinreleasing factor and galanin that originate mainly in the mesencephalic periaqueductal grey, the dorsal raphe nucleus and the peribrachial region of the dorsal pontine tegmentum. Brainstem

afferents

Posterior hypothalamus

Cholera toxin

SINCE von Economo’s clinical observations, a large corpus of experimental evidence indicates that the posterior hypothalamus has an important role in the maintenance of wakefulness. In fact, destruction or inactivation of neurons in the lateral part of the posterior hypothalamus results in somnolence or hypersomnia (2 1, 44, 49, 50, 57, 67, 68, 77, 89, 98). On the other hand, chemical or electrical stimulation of the posterior hypothalamus leads to the desynchronization of neocortical electroencephalogram (EEG) or to the states of arousal in normal as well as in “cerveau isolC” cats which exhibit a sustained synchronization of neocortical EEG (3, 30, 48, 87). It has been shown that the ventrolateral part of the posterior hypothalamus is conspicuous by the presence of magnocellular neurons and thereby called “caudal magnocellular” or “tuberomamillary” nucleus (TM) (6,7). Recent histochemical and immunohistochemical studies revealed that these magnocellular neurons contain histamine and constitute, with adjacent ventrolateral posterior hypothalamic neurons, the sole histaminergic cell group in the brain characterized by widely branching neuronal pathways influencing large target fields like noradrenergic and serotoninergic neurons of the lower brainstem (43,47,74,76, 102, 104, 115, 123). Single unit recording experiments suggested that these histaminergic neurons discharge tonically and especially during

Retrograde

transport

Double labeling

Cat

the periods of wakefulness (79,119). Recent pharmacological studies have also provided evidence for the important role of central histaminergic neurons in the regulation of states of vigilance (27, 48, 60, 70, 76). For example, in man and animals, systemic or intraventricular administration of antihistaminergic drugs induces drowsiness while histaminergic drugs cause wakefulness (48, 60, 70). Local injections of cY-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase, in the ventrolateral posterior hypothalamus increase deep slow wave sleep, while injections of SKF-91488, a specific inhibitor of histamine-Nmethyltransferase, or histamine itself in the same structure produce waking, and the waking effect induced by histamine is suppressed by pretreatment with mepyramine, a well-known histamine HI-receptor antagonist (48). A great deal of experimental evidence also pointed to a crucial role of several lower brainstem neuronal groups in the regulation of sleep and wakefulness such as ascending reticular neurons with still unknown neurotransmitters located in the mesencephalic, pontine and bulbar reticular formation (50, 63, 64, 10%107), serotoninergic neurons in the raphe nuclei, noradrenergic neurons in the locus coeruleus complex (24, 34, 38) and cholinergic neurons in the dorsal pontomesencephalic tegmentum (22, 39, 75, 84, 87, 99, 105). These findings, taken together, strongly suggest

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ABBREVIATIONS 3 3N 4 4N 5 5SP 6 7 7N 12 12N A T;Q AP BC BP CNF Cuti CS D EW F FTC GC

GCm GCP GR HLA IC ICA IN INC 10 IP K-F LC Ld& LI Lld LM LR LSC lvs MC

oculomotor nucleus oculomotor nerve trochlear nucleus trochlear nerve motor trigeminal nucleus alaminar spinal trigeminal nucleus, parvocellular division abducens nucleus facial nucleus facial nerve hypoglossal nucleus hypoglossal nerve nucleus ambiguus nucleus locus coeruleus, pars alpha aqueduct area postrema brachium conjunctivum brachium pontis nucleus cune~fo~is nucleus cuneatus nucleus raphe centralis superior nucleus of Darkschewitsch Ginger-Westphal nucleus fomix central tegmental field nucleus reticularis gigantocellularis griseum centrale mesencepbali griseum centrale pontis nucleus gracilis lateral hypothalamic area inferior colliculus interstitial nucleus of Cajal infundibular (arcuate) nucleus nucleus incertus inferior olivary complex interpeduncular nucleus Kiilliker-Fuse nucleus nucleus locus coeruleus nucleus ~ater~o~aIis tegmenti nucleus raphe linearis intermedius dorsal nucleus of the lateral lemniscus medial lemniscus nucleus raphe linearis rostralis and lateral reticular nucleus nucleus locus su~~~leus lateral vestibulospinal tract nucleus reticularis magnocellularis

MLB MT mV NTS P PAG PBG PbL

PbM PC

PCN peri-cu Pf PGCL PN :: PO0 PP PR PTM R RD RF

RM

Rob RP Rpa ztp SC SNC

SNL SNR so TD TM TV TB v3 VIN VM VMH X

medial longitudinal bundle mamillothalamic tract mesen~phal~c tract of t~gem~nus nucleus of the solitary tract pyramidal tract periaqueductal grey pa~bigeminal nucleus nucleus p~abrachialis lateralis nucleus pa~brachiaiis medialis nucleus reticularis parvocellularis nucleus of the posterior commissure nucleus peri-locus coeruleus alpha praedorsal bundle nucleus paragigantocellularis lateralis nucleus praepositus hypoglossi pedunculus mamillaris nucleus reticularis pontis caudalis nucleus reticularis pontis oralis ~~~duncular nucleus paramedian reticular nucleus medial pretectal nucleus red nucleus nucleus raphe dorsalis retroflex bundle nucleus raphe magnus nucleus raphe obscurus nucieus raphe pontis nucleus raphe pallidus rubrospinal tract nucleus reticularis tegmenti pontis superior colliculus substantia nigra, pars compacta substantia nigra, pars laterata substantia nigra, pars reticula&a superior olivary complex dorsal nucleus of Gudden tuberomamiliary nucleus ventral nucteus of Gudden trapezoid body third ventricle inferior vestibular nucleus medial vestibular nucleus ventromedial hypothala~c nucleus area X or nucleus tegmenti pedunculo~ntinus pars compacta

Nomenclature according to Berman (51, Nauta (691, Sakai (83.86) and Taber (113)

the existence of a substantial lower brainstem influence on the posterior hypothalamic neurons. In order to elucidate the brain mechanisms of sleep and waking, therefore, a prerequisite is to determine the brainstem afferents to the posterior h~th~amus and to identify their neu~~smitter characteristics. Although early anatomical studies with retrograde tracer techniques have been devoted to the search for neuronal connections from the lower brainstem structures to the posterior h~~~~us in the rat (4, 41, 42, 88, 116, 120), only little is known about the afferent projections or neurotransmitter contents of identified and nonidentified afferents to various parts of the cat posterior hypothalamus, such as the “histaminergic” ventrolateral (tuberomamillary nucleus, TM) and ~‘no~ist~nergic” dorsolateral posterior hypothalamus (lateral hy~~~a~c area, HLA). In the present study, therefore, using a double-immunostaining technique with unconjugated cholera toxin (CT) as a retrograde tracer (54,129, we investigated lower brainstem afferents to the various portions of the posterior h~thal~us and possible neu~~~smitters or

neurom~ulators

contained in these afferent connections in the cat. METHOD

Under ~ntob~bit~ anesthesia (25 mgikg, IV), 0.1 ~1 of a 1% solution of cholera toxin B subunit (CT) (List Biological Laboratories) was injected stereotaxically with a 5-p,l Hamilton syringe into the hypothalamus of seven adult cats, ranging from 2.5-4.0 kg in weight. Twenty-four hours after CT injections, the animals were treated with colchicine (200 kg in 20 pl of saline for each) by means of an injection cannula connected to a 50-~1 Hamilton syringe through guide cannulae previously implanted in the lateral and fourth ventricles. Twenty-four hours later, the cats were deeply ~es~etiz~ and perfused through the ascending aorta initially with 1,ooO ml of Ringer’s lactate solution and then with 2,500 ml of an ice-cold fixative containing 4% pamformaldehyde (PF), 0.05% glutaraldehyde and 0.2% 1-ethyl-3(3-dimethylaminopropyl) c~~dii~de (CD, Sigma) in 0.1 M phosphate buffer

BRAINSTEM AFFER.ENTS TO THE POSTERIOR HYPOTJL4LAMUS

,

‘. ,

PIG. 1. (A) Photomicrograph illustrating a cholera toxin (CT) injection site. (B-F) photomicrographs showing examples of CT-immunoreactive and tyrosine hydroxylase (B)-, phenylethanolamine N-methyltransferase (C)-, methionin-enkephalin-like (D)- or choline acetyltransferase (E, F)hnmunoreactive neurons located respectively in the nucleus raphe linearis (B), the caudal lateral medulla (C), the nucleus raphe dorsalis (D), the area X or nucleus mgmenti pedunculopontinus pars compacta (E) and the nucleus reticularis magnocellularis (P). Tbe double-labeled neurons are indicated by arrowheads. Small arrows denote neurons immunoreactive only to CT. Calibration bars = A, 1 mm; B-F, JO pm.

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ABOVE AND FOLLOWING PAGES FIG. 2. A series of camera lucida drawings of 22 J.L~sections from rostra1 (A) to caudal (U) order to illustrate the dis~bution of retrogradely labeled neurons after injections of cholera toxin in the ~~rorna~ll~ nucleus (Gl I l), the dorsolaterai part of the Posterior hypothalamus or the caudd part of the lateral hypothalamic area (H112) or the dorsomedial part of the posterior hypothalamus (R114). CT- and tyrosine hydroxylase (‘III)-immunoreactive double-labeled neurons observed in the caudal medulla are denoted by closed circles in P-U. Each dot and closed circle indicate, respectively, one retrogradely labeled and one double-labeled neuron.

(PB, pH 7.4). After removal from the cranium, brains were postfixed overnight in 0.1 M PB containing 2% PF and 0.5% CD at 4°C. The brains were then stored in 0.1 M PB containing 30% sucrose for 48-72 hours at 4°C. Sections of 22 pm thickness were cut in the frontal plane on a cryostat and then stocked in 0.1 M phosphate buffered saline (PBS) containing 0.3% Triton X-100 and 0.1% sodium azide (PBST-AZ, pH 7.4) before immunohistochemical staining. For immunohistochemical detection of CT, the sections were

first incubated in goat antiserum to CT (Vector Laboratories. dilution 1:40,000) for 2-4 days at 4°C. After several rinses, they were incubated overnight at 4°C in biotinylated anti-goat immuno~lobulin (Jackson I~unorese~ch Laboratories, 1:4$00). They were then rinsed and incubated in streptavidin conjugated with peroxidase (Jackson Immunoresearch Laboratories, 1:40,000) for 90 min at room temperature. After rinses, they were immersed with 0.025% 3,3’-diamino~nzidine-4 HCl (DAB, Sigma) containing 0.003% HZ02 and 0.6% nickel ammonium sulfate in 0.05

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FIG.2.CONTlNUED

M Tris-HCl buffer (pH 7.6) for IO-15 min at room temperature. For detection of neurotransmitter contents of CT-labeled neurons, the sections pretreated with CT immunohistochemistry were incubated in one of the following antibodies for 4-6 days at 4°C: 1) tyrosine hydroxylase (TH, Institute Jacques Boy, l:lO,OOO); 2) dopamine-B-hydroxylase (DBH, Eugene Tech International, 1: 5,000); 3) phenylethanolamine N-methyltransferase (PNMT, Eugene Tech International, 15,000 or a gift from Dr. L. Denoroy, Department of Experimental Medicine, France, l:lO,OOO); 4) serotonin (5HT, a gift from Dr. H. Kimura, Department of Anatomy, University of Shiga, Japan, 1:20,000); 5) methioninenkephalin (M-Et&, UCB, 1:5,000); 6) substance P (SP, Biogenex, 1:5,000); 7) corticotropin-releasing factor (CRF, UCB,

1:5,000); 8) galanin (Peninsula, 1:20,000). The specificity of each antibody against peptide was assessed by the absorption test. As to choline acetyltransferase (ChAT, Boehringer Mannheim, 1:2050) immunohistochemistry, the reaction was undertaken before CT immunohistochemical procedure. After several rinses, the sections were placed overnight at 4°C either in swine anti-rabbit IgG (DAKO, 1:800) or in biotinylated anti-mouse IgG (Vector Laboratories, 1:500). They were then rinsed and immersed either in rabbit PAP (DAKO, 1:800) or in avidin-biotin conjugated with peroxidase (Vector Laboratories) for 90 min at room temperature. After three rinses, the sections were reacted with 0.03% DAB containing 0.003% H,O, in 0.05 M Tris-HCl buffer for 15-20 min at room temperature. Finally, the sections were mounted on

SAKAI ETAL.

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t-i112

Gill

R 114

FIG. 2. ~O~INUED

gelatin-coated

slides,

dried,

dehydrated

and coverslipped

with

Depex. RESULTS

Description uf Injection Sites As described previously (25, 54,55, 125), injections of 0.1 ~1 of CT resulted in small deposits consisting of two distinct zones in the injection sites: a dense black deposit of the tracer at the center of the injection locus (central zone) and a surrounding lightly stained zone composed mainly of CT-labeled fibers (peripheral zone) (cf. Fig. IA). In one case (Gil l), the injection site was centered on the most ventrolateral part of the posterior hypothalamus largely confined to the tuberomamillary nucleus (TM) that contains the majority of histamine (HA)-immunoreactive cell bodies in the cat brain (47). In four cases (M105, N106, QllO and HI 12), injection sites were centered on the dorsolateral posterior h~~~arnus corresponding to the lateral hy~~~arnic area (HLA). In cases N106 and QllO, injection loci included the perifomical nucleus. In the remaining two animals, the injection loci were centered on the dorsomedial part of the posterior hypothalamus situated either just medial to the fomix and dorsal to the ventromedial hypothalamic nucleus (the dorsal part of the area

of the tuber cinereum) (R114) (Fig. 1A) or just lateral to the caudal part of the posterior hypothalamic area (B114). Three representative cases will be described in detail below (Fig. 2). Afferent Projections to the Posterior Hypothalamus Cholera toxin (CT) reaction products obtained by DAB-nickel histochemical procedures resulted in the presence of black punctate granules in retrogradely labeled neurons and fibers (Fig. 1). The localization of retrogradely labeled cells observed in three representative cases is depicted in Fig. 2A-U, showing camera lucida drawings of sections at different levels in the rostro-caudal extent of the lower brainstem. As illustrated in these figures, a substantial number of retrogradely labeled neurons was observed mostly after injections of the tracer in the dorsolateral posterior hypothalamus or caudal lateral hypothalamic area. As a whole, the lower brainstem afferents were predominantly ipsilateral. Midbrain. There was a large number of small to medium-sized CT-iabeled neurons in the entire extent of the ~~~uedu~l grey (PAG) and the mesencephalic reticular formation or central tegmental field (FTC). In addition, the ventral tegmental area (VTA), the substantia nigra (SN) and adjacent retrorubral nucleus (RR), as well as the medial pretectal nucleus (PTM), contained many retrogradely labeled cells particularly after CT injections

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0 CT +TH

FIG. 2. CONTINUED

into the dorsolateral part of the posterior hypothalamus (case H112) (Fig. 2A-D). Similarly, after CT injections into the dorsolateral posterior hypothalamus, we noted many CT-labeled neurons in the nucleus raphe linearis intermedius (LI) and a moderate number of cells in the Edinger-Westphal nucleus (E-W) (Fig. 2A, B). On the other hand, the peripeduncular nucleus (PP) contained numerous retrogradely labeled cells in all injection cases (Fig. 2A-C). Pans. As in the midbrain, following injections of the tracer in the dorsolateral posterior hypothalamus, a great number of retrogradely labeled, small to medium-sized cells was found in the periaqueductal grey (PAG) and in the rostra1 pontine reticular formation (Poo). Numerous CT-labeled neurons were also observed in the following pontine structures (Fig. 2E-H): 1) the nucleus tegmenti pedunculopontinus pars compacta (69) or X area (83); 2) a paralemniscal region; 3) the parabrachial nuclei; 4) the peri-locus coeruleus alpha (perk); 5) the locus coeruleus complex consisting of the nucleus locus coeruleus (LC), locus coeruleus

alpha (LCa) and locus subcoeruleus (LSC); 6) the nucleus laterodorsalis tegmenti (Ldt); and 7) the nuclei raphe dorsalis (RD) and centralis superior (CS). A moderate number of retrogradely labeled cells was found in the nucleus incertus (INC) (Fig. 21-K), the rostra1 part of the nucleus raphe magnus (RM) (Fig. 2J-M), the caudal part of the nucleus reticula& pontis oralis (Poo) and the nucleus reticularis pontis caudalis (Pot) (Fig. 2E-K). The labeled neurons in the RM were mostly medium-sized, whereas those in the other structures were mainly small. The nucleus cuneiformis (CNF) contained a few CT-labeled small cells (Fig. 2G, H). Medulla. At the level of the rostral medulla, a moderate number of retrogradely labeled neurons was found in the nucleus reticularis magnocellularis (MC) almost exclusively after injections of the tracer in the dorsolateral posterior hypothalamus. These labeled neurons were fusiform or multipolar and medium-sized to large. A few CT-labeled, small to medium-sized cells were also scattered in the nuclei reticularis gigantocellularis (Gc) and par-

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FIG. 3. Pbotomicrographsto illustrate tyrosine hydroxylase (A)-, serotonin (B)-, choline acetyltransferase(C)- or methionin-enkephalin-like(D)immunoreactivenerve tibers observed in the caudal part of the lateral h~pothaIamicareas. Calibration bars= A-D. 50 p.m.

vocellularis (PC) (Fig. 2L-R). At the level of the caudal medulla, a few CT-labeled cells were observed, in all injection cases, in the caudal lateral bulbar reticular fo~ation around the nucleus ambiguus (A) and lateral reticular nucleus (LR), and in and just around the nucleus of the solitary tract (NTS) as well. These neurons were mainly medium-sized and distributed bilaterally (Fig. ZP-U). ImmunohistochemicaL Nature of the Brainstem A&rents Catecholaminergic inputs. As shown in Fig. 3A, in addition to densely labeled TH-immunoreactive fiber bundles observed in the most dorsal part of the lateral hy~~alamus, a moderate to high density of TH-immunoreactive fibers was observed in the lateral hypothalamic area. Dopaminergic inputs. After CT injections in the dorsolateral posterior hypothalamus, many retrogradely labeled neurons immunoreactive to tyrosine hydroxylase (TH) [but not to dop~ineP-hydroxylase (DBH)] were found in the pars compacta (SNC) of the substantia nigra [A9 cell group of Dahlstrtim and Fuxe (I@], the ventral tegmental area of Tsai (VTA) (AlO) and a medial lemniscal area composing the retrombral nucleus (RR) (A@. In sharp contrast, virtually no double-labeled cells were found in these structures following CT injections in the tuberomamiliary

nucleus (TM) or dorsomedial posterior hypothalamus (Figs. 2A-D and 4). Some CT- and TH-immunoreactive double-labeled neurons were also found in the nucleus raphe inte~edius (LI) (AlO) (Fig. 3Bf and in the rostra1 part of the dorsal raphe nucleus (RD) [All of Lindvall and Bjtirklund @I)]. The latter double-labeled cells were found especially after CT injections into the dorsomedial posterior hy~~alamus (Fig. 6A). ~oradrenerg~c inpufs. As shown in Fig. 6, only scattered CT- and tyrosine hydroxylase (TH)-positive double-labeled neurons were found in the dorsal pontine tegmentum such as the nuclei locus coeruleus (LC) (A@, locus coeruleus pars alpha (Lea) (A6), locus su~oe~leus (LSC) (A7). nuclei p~abrachialis lateralis (PbL) and medialis (PbM) (A7) and KGlliker-Fuse nucleus (K-F) (A7). These neurons were mostly medium-sized. The tracer injections in the TM or dorsomedial part of the posterior hypothalamus resulted in many fewer double-labeled cells than those in the dorsolateral part of the posterior hypo~al~us. A4 and A5 noradrenergic cell groups were never double Iabeled. Although few in number, most of retrogradely labeled neurons in the caudal medulla, particularly those located ipsilaterally to the injection sites, showed TH-like immunoreactivity (Fig. 2P-U). After CT injections in the dorsomedial part of the posterior hypothalamus, the great majority of retrogradely labeled neurons

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HI.12

FIG. 4. (A-D) Camera luckla drawings of ~p~~n~tive sections depicting cholera toxin (CT)positive and serotonin (S-I-IT) (open squares)- or tyrosine hydroxylase (TH) (closed circles)immunoreactive double-labeled neurons observed after a CT injection in the dorsolateral posterior hypothalamus. (a-c) higher magnifications of the boxed areas in A-C to illustrate the localization of neurons irmnunoreactive only to CT (open circles) and that of CT- and TH-immunoreactive double-labeled neurons.

within and around the nucleus of the solitary tract (NTS) were immunoreactive to TH and dopamine-B-hydroxylase (DBH), but not to phenylethanolamine N-methyltransferase (PNMT) (A2 cell group). Similarly, CT-labeled cells in the most caudal part of the ventral medulla (Fig. 2T, U) were immunoreactive to TH and DBH, but not to PNMT, thus being a member of Al cell group. In contrast, the vast majority (8~~%) of CT- and TH-positive double-labeled cells observed in the rostral part of the lateral medulla (Fig. 2Q-S) showed PNMT-like immunoreactivity (Cl cell group) (Fig. 10 Serotoninergic inputs. A high density of serotonin (5HT)immunoreactive fibers was found in the entire posterior hypothalamus (Fig. 3B). CT-positive and 5-HT-i~uno~active double-ladled neurons were seen in the nuclei raphe intermedius (LI) [B8 cell group (16), Figs. 4B-D and SA], dorsalis (RD) (B7), centralis superior (CS) (B8), as well as within and around the nucleus incertus (INC) (B6) (Fig. 5). A few double-labeled cells were also noted in the dorsal pontine tegmentum lateral to me raphe nuclei (Fig. 5). CT

injections in the dorsolateral posterior hypothalamus resulted in the double-labeling of neurons in the entire extent of the dorsal raphe nucleus, whereas injections in the tuberomamillary nucleus (TM) or dorsomedial posterior hypothalamus led to double-labeled neurons mostly in the caudal part of the nucleus. Many (2&50%) neurons were doubly labeled in the RD, whereas only a few (less than 10%) were doubly stained in the CS in spite of a large number of retrogradely labeled neurons in the latter nucleus (Fig. 5b, c). No double-labeled cells were identified in other raphe nuclei such as the nuclei raphe pontis (B5), magnus (B3) and pallidus (Bl). Cholinergic inputs. As shown in Fig. 3C, tine ChAT-immunoreactive fibers were observed in the caudal lateral hypothalamic area. As shown in Fig. 6, there were few or no CT-positive and choline acetyltransferase (ChAT)-immunoreactive double-labeled neurons in the lower brainstem cholinergic structures, when CT was injected either into the TM (case Gl 11) or into the dorsomedial posterior hypothalamus (case R114). However, a relatively large number of double-labeled neurons were observed in the

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HI12 If

v.' a

R114

H 112

l

CTiSHT

FIG. 5. Locations of cholera toxin (CT)- and serotonin (5-HT)-immunoreactive double-stained neurons (closed circles) detected after CT injection in the tuberomamillary nucleus (Gl 1I), the dorsolateral (H112) or dorsomediaf posterior hypothalamus (R114). Each symbol indicates one double-labeled cell noted on one representative section. (a-c) camera lucida drawings of representative sections to show the exact localization of CT- and SHTimmunoreactive double-labeled neurons (closed circles), as well as those immunoreactive only to CT (open circles) in case H112.

dorsal pontine tegmental cholinergic structures following injections of the tracer into the dorsolateral posterior h~thalamus (case HI 12) (Fig. 6). They were located mostly within and just

around the brachium conjunctivum (BC) at the level of the caudal two-thirds of the ~dunculo~ntine tegmental nucleus (69) or X area (83) (X), the rostra1 part of the nucleus peri-locus coeruleus

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FIG. 6. Locationsofcholeratoxin (CT)-positiveand choline acetyltransferase(ChAT) (open circles)-or tyrosine hydroxylase (TH) (closed circles)-immunoreactive double-labeled neurons projecting directly to the tuberomamillary nucleus (Gl 1l), the dorsolateral (HI 12) or dorsomedial fRl14) posterior hypothalamus. Each symbol indicates one double-ladled neuron observed on one ~p~~n~tive section. Arrows at P3 denote CT- and BAT-i~uno~active double-stained small cells.

alpha (‘t-i-o) and the nuclei p~brachialis lateralis (FbL) and medialis (PbM). A few double-labeled neurons were also found in the nuclei laterodorsalis tegmenti (Ldt), locus coeruleus alpha (LCa) and locus subcoeruleus (LSC). These double-labeled neurons represented only 510% of total retrogradely labeled neurons in the same areas (Fig. 7-l). It should be noted, however, that the great majority of only retrogradely labeled neurons were small (7-12 x 12-20 pm), whereas all double-labeled neurons were medium-sized to large (12-18 x 20-30 pm) except for some double-labeled cells in the most caudal part of the LCa and PbM that were composed of oval, small cells (7-12 x 16-20 Pm) (Fig. 6, P3 arrows). A group of the largest cholinergic neurons (18-32 x 32-46 pm) was never double stained (Fig. 1E). In addition to these pontine cholinergic neurons, there was a ~p~ation of medium- to large-sized (14-25 x 30-42 cm) doublelabeled neurons in the middle and lateral part of the nucleus

reticular& magn~ell~~s (MC) of the ventral medulla. These neurons were observed especially after CT injections into the dorsolateral posterior hypothalamus (Figs. 1F and 7-2). PeptideFgic inputs. The results obtained from three representative cases are depicted in Fig. 8. Like the monoaminergic and cholinergic afferent projections, CT injections in the dorsolateral part of the posterior hypothalamus resulted in more numerous double-labeled neurons. Among the neuropeptides investigated, there were many more double-labeled neurons with methioninenkephalin (M-Et&) than with substance P (SP) and corticotropinreleasing factor (Cl@). Virtually no galanin-like inputs were identified. It should be mentioned that a high density of MEnk-like immunoreactive fibers was observed in the lateral posterior hypothalamus (Fig. 3D). On the whole, these double-labeled neurons were similarly localized and most of them were observed in: 1) mesencephalic periaqueductal grey (PAG); 2) dorsal raphe

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DISCUSSION

3.

The present findings indicate that neurons in the dorsolateral part of the posterior hypothalamus, as compared with those in the ventrolateral or dorsomedial parts, receive substantial and widespread projections from lower brainstem neurons that contain dcetylcholine, serotonin, dopamine, noradrenaline, adrenaline, neuropeptides such as methionin-enkephalin (M-Enk) and, to a lesser extent, substance P (SP) and corticotropin-releasing factor (CRfl, and some unknown neurotransmitters or neuromodulators. It is well known, however, that the medial forebrain bundle fills practically the entire lateral hy~thalamus (71,73) and the question arises as to whether the present retrograde tracing data are due to the uptake by axon terminals or uptake by fibers of passage. We reported previously that, in contrast to free- or WGA-conjugated HRP, there is little uptake of CT by fibers of passage (54,55). In line with these reports, our CT injections in the preoptic region of the lateral hypothalamic area resulted in far fewer retrogradely labeled neurons throughout the lower brainstem structures as compared to those found after injections in the posterior hypothalamus (our unpublished data). These observations suggest that the present retrograde-labeling data are largely ascribable to the uptake and subsequent retrograde transport of CT which has been shown to have a high binding capacity to monosialoganglioside receptors on the nerve terminal surface (95,109). A series of early studies in the rat using HRP retrograde transport techniques also provided evidence sup~~ing the ~sumption that the major ascending components of the medial forebrain bundle originating in the lower brainstem terminate at the level of the posterior hypothalamus (41, 42, 88, 116, 120). Nevertheless, we cannot rule out the possibility of uptake and subsequent retrograde transport of the tracer by axons of passage that were undamaged or damaged during the injection procedures. We will therefore first compare our retrograde-labeling data with those of previous retrograde, as well as anterograde transport studies using autoradiographic or phaseolus vulgaris leucoagglutinin (PHA-L) tracing methods. We will then consider their immunohistochemical characterization.

FIG. 7. Camera iucida drawings of frontal sections of the left dorsal pens [1] and rostra1 medulla [Z] to itlustrate the localization of cholera toxin (CT)-positive aione (open circles), choline acetyltransferase (ChATfimmunoreactive alone (dots) or CT-positive and ChAT-immunoreactive double-stained neurons (closed circles) that send axons directly COthe dorsolateral posterior hypothalamus. Each symbol indicates one doublelabeled neuron on one representative

section.

nucleus (RD); 3) a lateral marginal region of the brachium conjunctivum (BC) comprising the nuclei parabrachialis lateralis (PbL) and locus subcoeruleus (LSC); and 4) a lemniscal region situated dorsomedial to the brachium pontis (BP). In addition, the nucleus laterodorsalis tegmenti (Ldt) and neighboring locus coeruleus (LC) and locus coeruleus CL(LCCX)contained CT-labeled neurons that were immunoreactive to M-E& whereas those immunoreactive to SP were also located within and around the nucleus incertus (INC). As a whole, the double-labeled peptidergic neurons were small and round or oval, but, like 5HT-immunoreactive neurons, CTand M-Enk-immunoreactive double-labeled neurons in the RD were medium-sized and multipolar (Fig. 1D) and represented 20-50% of total retrogradely labeled neurons in this nucleus (Fig. 8, HI12 a-c). A few CT- and M-Enk-immunoreactive doublelabeled neurons in the LC complex were mostly medium-sized and they seemed to colocalize with noradrenergic neurons.

Afferent prQjec~i~ns~~~ the midbrain. Previous studies in the rat reported the presence of a large number of retrogradely labeled cells in many of the same lower brainstem structures as shown in the present study, e.g., mesencephalic and rostral pontine reticular formation (RF) and periaqueductal grey (PAG), peripeduncular nucleus (PP), rostra1 raphe nuclei, and parabrachial regions of the pontine tegmentum (4, 41, 42, 116, 120). The present study in the cat confirms previous findings obtained in the rat showing large and rather specific afferent projections from the midbrain RF to the dorsolateral posterior hypothalamus, as well as moderate and diffuse projections from the more laterally located ~~~duncul~ nucleus (PP) to the whole posterior hy~th~~us. In a recent autoradio~aphic study in the rat, Eberhart ei al. (20) reported that a moderate number of ascending fibers originating in the mesencephalic RF terminate in the caudal lateral hypothalamus. Ascending projections from the central periaqueductal grey matter (PAG) to the posterior hypothalamic area were fist demonstrated by silver degeneration technique (11,29). The present study revealed the existence of a diffuse ascending projection from the entire extent of the PAG to the posterior hypothalamic structures and, therefore, does not confirm an early suggestion that the cat PAG is composed to distinct regions, each having distinct efferent connections (29). Recent autoradiographic studies in the

BRAINSTEM

APEXRENTS TO THE POSTERIOR

G 111

l

M-Enk

HYPOTHALAMUS

H 112

h SP

A CRF

R 114

* Galanin

FIG. 8. Locations of cholera toxin (CT)-positive and methionin-enkephalin-like (M-Enk) (closed circles)-, substance P-like (SP) (open triangles)-, corticotropin-releasing factor-like (CW) (closed triangles)- or galanin-like (asterisks)-immunoreactive double-stained cell bodies. (a-d) Camera lucida drawings of representative sections showing the localization of CT-positive alone (open circles) and CT-positive and M-Enk-immunoreactive double-labeled neurons (closed circles) in the dorsal raphe nucleus (a-c) and in a peribrachial region of the dorsal pontine tegmentum (d) observed in case H112. Each symbol represents one double-stained cell detected on one representative section. (20) and monkey (58) also described a diffuse ascending projection from the PAG to the hypothalamus, in particular its lateral part. A heavy innervation of the caudal lateral hypothalamus by the ventral tegmental area (VTA) and adjacent substantia nigra (SN)

rat

as demonstrated in the present study is at variance with an HRP study in the rat (4), but consistent with other retrograde transport studies in the same animal (42, 116, 120). Previous retrograde studies also reported, however, that neurons in the VTA and the pars compacta of the SN (SNC) send axons directly to the anterior

450

hypothalamic areas through the medial forebrain bundle (37, 96, 115, 120). In addition, early autora~ographic studies failed to determine whether or not any VTA and SN fibers terminate along the bundles running through the lateral hypothalamus (2,101). It seems likely that the bundle terminates mainly in the posterior hypothalamus, since CT injections in the preoptic regions of the cat anterior hy~thalamus result in very few retrogradely labeled cells in the VTA and SN (our unpublished data). However, we cannot eliminate the possibility of uptake of CT by fibers of passage, since the ascending fibers might terminate at the tuberal or anterior hypothalamic level. Further studies are thus required to clarify this problem. A possible inne~ation of the h~thalamus by mesencephalic dopamine neurons will be discussed below. Agerent projectionsfiom the pons. Projections from the rostra1 raphe nuclei to the hypothalamus have been reported by many authors (1, 4, 10, 13, 41, 42, 61, 94, 100, 116, 120). In the present study, the tracer injections in the dorsolateral posterior hypothalamus led to many more retrogradely labeled cells in the rostra1 raphe nuclei than those in the medial or ventrolateral (tuberomamillary nucleus, TM) part of the posterior hypothalamus, Our findings are in agreement with a previous observation made by Berk and Finkeistein (4) in the rat. The present findings in the cat confirm recent retrograde-labeling data demonstrating ascending projections from the rostral raphe nuclei to the rat TM (23). Although CT-labeied neurons were identified in all cases throughout the rostrocaudal extent of the nucleus raphe dorsalis (RD), our findings reveal heavy projections from the rostra1 part of the RD to the dorsolateral part of the posterior hypothalamus. In contrast, the TM appears to receive heavier projections from the caudal part of the RD. Our observations are thus in line with an earlier retrograde study showing the existence of differential ascending projections of neurons within the RD projecting either to the amygdala-piriform cortex and hippocampus or to the striatum and neocortex (33). In the present study, CT injections in the posterior hypothalamus, especially its dorsolateral part, resulted in retrograde labeling of neurons in the raphe magnus and, to a much lesser exent, the rostra1 part of the raphe pallidus, but few or none in the nuclei raphe pontis and obscurus. InneNation of the hy~th~~us by raphe magnus neurons has been demonstrated in the rat and cat with HRP and autoradiographic methods (4, 10, 42, 116, 120). Innervation of the hypothalamus by neurons of the raphe pontis, p~lidus and obscurus, however, is still a controversial issue. Several retrograde tracing studies reported in the rat the existence of ascending projections from the raphe pontis and obscurus to the posterolateral hypothalamus (4, 116, 120), but not all studies support this view (4). In the present study, retrogradely labeled cells were noted around the raphe pontis, but the raphe pontis itself did not contain these cells. Similarly, some labeled cells were found in the midline raphe located dorsally to the raphe magnus, but we did not find retrogradely labeled cells in the raphe obscurus. These discrepancies might be due to species differences. In keeping with early anterograde and retrograde studies (4,42, 56, 90, 116), parabrachial (PB) neurons appeared to project extensively to the entire posterior hypothalamus in the cat. Our data are also in accord with early observations of Saper and Loewy (90) reporting that the PB-h~th~amic projection originates primarily from the lateral parabrachial nucleus (PbL) and that the projection to the lateral hypothalamic area is the densest at the level of the posterior hypothalamus. Ascending projections from the locus coeruleus will be discussed below. In the present study, both dorsal and ventral (or deep) tegmental nuclei of Gudden were devoid essentially of retrogradely labeled neurons, though a significant number of labeled cells was found around these nuclei after CT injections in the posterior

SAKAI ET AL.

hypothalamus including the tuberomamillary nucleus (TM). In a recent paper, Ericson ef at. (23) showed the presence of afferent projections to the rat TM arising from both the dorsal and ventral nuclei of Gudden. They mentioned that numerous retrogradely labeled cells were found in the dorsal tegmental nucleus of Gudden after injections of the fluorescent dye Fast Blue in the ventral subgroup of the TM located near the lateral mamillary nucleus, but not after injections of the tracer in the medial subgroup of the TM located near the dorsal premamillary nucleus. In contrast, the ventral and dorsal tegmental nuclei of Gudden have been reported to contain a large number of retrogradely labeled cells after injections of Fast Blue in the medial subgroup of the TM. According to the same authors, injection sites involved either a part of the lateral mamillary nucleus or the dorsal and ventral premamillary nuclei. Recently, using WGA-HRP anterograde and retrograde tracing methods, Shibata (97) demons~ated ascending projections from the dorsal tegmental nucleus to the lateral mamillary nucleus and from the ventral tegmental nucleus to the medial mamillary and premamillary nuclei. From these and other early anatomical findings, it appears that there are in fact no direct projections from the dorsal and ventral tegmental nuclei of Gudden to the TM in the rat and cat. ABrent projections from the medulla. In the medulla, the rostra1 part of the nucleus reticularis magnocellularis (MC) (or magnocellular tegmental field in the cat or nucleus reticularis gigantocellul~s pars alpha in the rat brain) contained a significant number of retrogradely labeled neurons particularly following injections of the tracer into the dorsolateral posterior hypothalamus. These ascending neurons were scattered, however, within the MC. This might explain why some early retrograde tracing studies in the rat failed to demonstrate the Mc-h~o~alamic projection (4, 41.42). Recent anterograde and retrograde transport studies, however, have evidenced the existence of this ascending projection in the rat brain (35,121). The inneNation of the posterior h~~al~us by the caudal lateral bulbar reticular formation has been reported by many previous authors (4, 23, 53, 88, 116). In the present study, we found a diffuse ascending projection from the caudal bulbar reticular fo~ation to the posterior hy~thalamus, but the major component of the ascending fibers appeared to pass through or terminate in the dorsal or dorsomedial part of the posterior hypothalamus. This is consistent with the early anatomical findings. Using autoradio~aphic anterograde tracing methods, Ricardo and Koh (81) and Ciriello and Calaresu (12) have described ascending projections from the nucleus of the solitary tract (NTS) to the hypothalamus. These authors reported a sparse and diffuse labeling in the lateral, in particular dorsolateral, part of the posterior hypo~~~us, whereas a heavier labeling was noted in the dorsomedial region of the posterior hypothalamus. In keeping with these observations, the greatest number of retrogradely labeled neurons was identified in the caudal part of the NTS after CT injections in the dorsomedial posterior h~o~alamus. The NTS also contained some CT-labeled neurons after injections of the tracer in the dorsolateral posterior hypothalamus, but none following injection in the tuberomamillary nucleus (TM). These findings will be further discussed below with reference to catecholaminergic afferent projections. Immunohistochemical Properties of the Brainstem Afferents ~atechoiaminergic afferents. ~opaminergic inputs. The rat posterior hy~~~~us has been shown to contain TH-, DBH-, PNMT-, as well as fi-HT-immunoreactive (IR) fibers (23, 31, 32, 103, 112). Using electron microscope immunohistochemic~ technique, Ericson et al. (23)

BRAINSTEM

AFFERENTS

TO THE POSTERIOR

HYPOTHALAMUS

have demonstrated recently the terminations of axons immunoreactive to TH, DBH or 5-HT in the rat tuberomamillary nucleus (TM) that contains histamine (HA)-IR neurons. In the present study in the cat brain, we also observed dense TH-IR varicose fibers in the caudal lateral hypothalamic area and in the tuberomamillary nucleus (TM) as well. We observed numerous CT- and TH-IR double-labeled neurons in the substantia nigra (SN) and ventral tegmental area (VTA) following injections of the tracer in the dorsolateral posterior hypothalamus, but identified virtually no double-labeled neurons in these ventral tegmental structures after injections in the tuberomamillary nucleus (TM) nor in the dorsomedial posterior hypothalamus. As mentioned above, the innervation of the posterior hypothalamus by the mesencephalic dopamine neurons is still a matter of debate. However, it seems likely that the mesencephalic dopaminergic neurons give rise to ascending axons passing through and terminating in the posterior hypothalamus, especially in its dorsolateral part, since CT injections in the preoptic region of the medial forebrain bundle produced far fewer CT- and TH-IR double-labeled neurons in the mesencephalic dopamine cell groups as compared with CT injections in the posterior hypothalamus (our unpublished data). In a previous paper, we provided evidence for the dopaminergic innervation of the whole posterior hypothalamus by neurons intrinsic to the hypothalamus such as the Al 1 and Al3 dopamine cell groups (125). Taken together, it can be concluded that the major dopaminergic innervation of the TM originates not in the mesencephalit dopamine cell groups (A8, A9 and AlO), but in the hypothalamic dopamine cell groups. According to our study, dopamine neurons in the nucleus raphe intermedius (LI) (AlO) appear to innervate the dorsolateral posterior hypothalamus, but those located in the rostra1 part of the dorsal raphe nucleus (RD) [All cell group (51,72)] do not. Instead, these Al 1 dopamine neurons seem to project directly and selectively to the dorsomedial posterior hypothalamus. Our findings thus extend earlier observations in the rat on the ascending projections from the Al 1 dopamine neurons to the striatum (18) and hippocampus (80). Noradrenergic inputs. Using HRP retrograde tracer techniques combined with either monoamine oxidase staining or catecholamine fluorescence, earlier studies demonstrated in the rat and rabbit the catecholaminergic innervation of the preoptic and anterior hypothalamus by pontine (A6) and medullary (Al and A2) cell groups (8, 17, 23, 88). It has also been suggested that in the rat the DBH-positive cells located in the ventrolateral and dorsomedial medulla with the involvement of the locus coeruleus neurons represent the major sources of noradrenergic afferents from the lower brainstem to the hypothalamus (62,93). The present study confiied these findings and further showed that in the cat: 1) the great majority of CT- and TH-positive doublelabeled neurons in the rostral medulla (that contains clusters of both adrenergic and noradrenergic neurons) corresponds to those immunoreactive to PNMT or adrenergic neurons; 2) a few Al noradrenergic neurons in the caudal medulla project to the whole posterior hypothalamus; 3) A2 noradrenergic neurons send ascending axons especially to the dorsomedial part of the posterior hypothalamus; and 4) noradrenergic neurons in the dorsolateral pontine tegmentum (A6 and A7) innervate particularly the dorsolateral part of the posterior hypothalamus. Adrenergic inputs. Using phenylethanolamine N-methyltransferase (PNMT) immunohistochemistry, H&felt et al. (31) first demonstrated in the rat a modest adrenergic innervation of the posterior hypothalamus. It has also been reported that the rat hypothalamus receives a diffuse adrenergic innervation by medullary adrenergic cell group such as the Cl, C2 and C3 (23, 82, 91, 117). Although the presence of PNMT-immunoreactive fibers has not yet been demonstrated in the region of the cat posterior hypothalamus, the

451

present study provides evidence for the presence of a diffuse adrenergic innervation of the cat posterior hypothalamus by a medullary adrenergic cell group that represents the feline homologue of Cl as designated by H&felt et al. (31) in the rat brain. The present study confiis a recent report by Ericson et al. (23) on the adrenergic innervation of the rat tuberomamillary nucleus by the medullary adrenergic neurons, even though these ascending adrenergic projections do not seem to be very strong in the cat as compared with those projecting to other regions of the posterior hypothalamus. Direct projections from the adrenergic neurons to the magnocellular basal forebrain have been reported in the rat (96). However, our recent double-labeling study showed that the adrenergic innervation of the preoptic and basal forebrain areas was very sparse in the cat (our unpublished data). Serotoninergic afferents. The existence of a dense 5-HT innervation of the entire posterior hypothalamus is well known [see (103,118)]. In line with early observations, CT injections in the lateral hypothalamus led to many more retrogradely labeled neurons in the nuclei raphe dorsalis (RD) and centralis superior (CS) than injections in the medial part of the hypothalamus. The present double-labeling data further indicate that major ascending serotoninergic projections to the cat posterior hypothalamus, in particular its dorsolateral part, arise from serotoninergic cell groups located in the entire rostrocaudal extent of the RD and the dorsal part of the CS, designated, respectively, B7 and B8 by Dahlstriim and Fuxe (16). In addition, minor serotoninergic afferents derive from B6 and B8 cell groups located, respectively, in the nucleus incertus (INC) and the nucleus raphe linearis intermedius (LI). Our findings also revealed scarce ascending projections to the cat posterior hypothalamus from B9 serotonin cell groups located among or near the fibers of the medial lemniscus and none from the B5 cell group situated in the nucleus raphe pontis. As discussed above, however, the B5 cell group appears to send axons to the posterior hypothalamus in the rat. This discrepancy regarding B5 ascending projections in the rat and cat might be due to species differences. Following injections of CT in the tuberomamillaty nucleus (TM) or dorsomedial posterior hypothalamus, we found relatively many CT-positive and 5-HT-immunoreactive double-labeled neurons in the caudal part of the RD, though numerous retrogradely labeled neurons were found throughout the rostrocaudal extent of the nucleus. Our findings in the cat are consistent with those in the rat reported recently by Ericson et al. (23), even though they did not mention any difference in the distribution of the ascending serotoninergic neurons within the dorsal raphe nucleus. Cholinergic aflerents. Early immunohistochemical studies using choline acetyltransferase (ChAT) as a marker have evoked a cholinergic innervation of the entire hypothalamus, especially the lateral part (40, 78, 114). In a previous paper, we demonstrated that the cat posterior hypothalamus receives only a slight cholinergic afferent projection arising from the preoptic-basal forebrain structures such as the septum, the lateral preoptic area and the nuclei of the diagonal band of Broca (125). It appears therefore that the major cholinergic afferents to the cat posterior hypothalamus originate in the lower brainstem (36, 86, 122). Early autoradiographic or PI-IA-L anterograde studies demonstrated the direct ascending projections from the pedunculopontine tegmental nucleus (or X area) and nucleus laterodorsalis tegmenti (Ldt) to the posterior hypothalamus in the rat and cat (26, 92, 110) and the cholinergic property of the ascending tegmental neurons was further suggested by the results from retrograde transport study of [3H]choline (110). Using fluorescent tracer histology combined with ChAT immunohistochemistry or acetylcholinesterase (AChE) pharmacohistochemistry, Woolf and Butcher (124) have revealed the cholinergic innervation of the rat lateral hypothalamus by the pedunculopontine and lateral dorsal tegmental nuclei. The present

SAKAI ET AL.

452

study confirmed these findings and further revealed that, in the cat, the ascending pontine cholinergic neurons are located mostly within and just around the brachium conjunctivum (BC) at the level of the caudal two-thirds of the X area and neighbouring parabrachial nuclei (PbL, PbM), and, to a lesser extent, in the nuclei per&locus coeruleus (Y(peri-o) locus coeruleus a (LCa), locus subcoeruleus (LSC) and laterodorsalis tegmenti (Ldt). Except for a few small double-labeled cells in the most caudal part of the LCa and PbM, these ascending cholinergic tegmental neurons were medium-sized to large, and a group of the largest cholinergic neurons was never double labeled. In contrast, early studies reported in the cat that the tegmental cholinergic neurons projecting directly to the thalamus are located mainly in the rostra1 two-thirds of the X area, in particular within and dorsal to the BC, as we11as in the pet-i-L&, the rostra1 part of the L&Y and the Ldt. These neurons were reported to be m~ium-sized to very large (19, 28, 75, 83, 85, 108, 124). On the whole, these anatomical findings suggest differences in the localization and morphology between the tegmental cholinergic neurons sending axons to the posterior hypothalamus and those projecting directly to the thalamic structures. The problem as to a possible bifurcating cholinergic projection from the pontine tegmental neurons to the thalamus and hy~~al~us is currently being investigated in our laboratory. The present study evidenced for the first time the direct ascending cholinergic projection to the posterior hypothalamus originating in the nucleus reticularis magnocellularis (MC) of the medulla oblongata, which corresponds to the most caudal part of the ascending reticular activating system of Moruzzi and Magoun (64) and Lindsley er al. (50). These ascending cholinergic neurons were medium-sized to large and they occupied the most lateral part of the middle portion of the MC, being within and just medial to the direct lateral vestibulospinal fibers. At present, there is no report on the direct cholinergic projection from the medulla to the thalamus. Pepr~~ergic ujjferents. The posterior hy~~al~us has been reported to contain many peptidergic nerve terminals (9, 15, 32, 52.59). In a previous study, we demonstrated that the cat posterior hypothalamus in general receives peptidergic afferents such as methionin-enkephalin (M-Et&), galanin, neurotensin (NT), substance P (SP) and corticotropin-releasing factor (CRF) that arise from the medial preoptic area, the nucleus of the stria terminalis and/or the posterior hy~~~a~c structures (125). In the present study, we found that a number of retrogradely labeled neurons in the periaqueductal grey (PAG), dorsal raphe nucleus (RD) and

dorsal pontine tegmentum were i~unoreactive to M-E& substance P (SP), CRF and, to a much lesser extent, to galanin, when CT was injected into the dorsolateral posterior hypothalamus. Our results are in accord with earlier observations on the existence of dense SP-like, M-Et&-like, and CRF-like nerve terminals in the dorsolateral part of the rat posterior hypothalamus (9, 15, 32). The existence of neuronal perikarya immunoreactive to SP, M-Enk or CRF has been reported in the ~~aqueduct~ grey (PAG), nucleus raphe dorsalis (RD) and dorsal pontine tegmentum of the rat and cat brain (14, 45, 46, 65, 66, 111). The descending projection from these tegmental peptidergic neurons to the nucleus reticularis magnocellularis (MC) has been demonstrated recently in the cat (55). In general, the double-labeled peptidergic neurons were small, but, like serotonin (5-~)-immunoreactive neurons, CT- and M-Et&-like immunoreactive neurons in the RD were medium and, like CT- and S-HT-immunoreactive double-labeled neurons, they represented 20-50% of all the retrogradely labeled neurons in this nucleus. These results suggest a possible colocalization of 5-HT and M-Enk in the same RD neurons sending axons to the posterior hypothalamus. However, this seems unlikely since their localization within the RD was not exactly the same, and since the colocalization of M-Enk and S-HT in the dorsal raphe neurons has been reported to be almost negligible in the cat brain (46). In contrast, most of the medium-sized retrogradely labeled neurons in the LC complex appeared to exhibit both TH- and M-Enk immunoreactivity. The colocalization of noradrenaline and M-Enk in the locus coeruleus complex neurons has been reported in the cat by Uger et af. (45). In conclusion, the present study provides evidence that the dorsolateral part of the posterior hypothalamus, which has an important role in the regulation of the states of vigilance, receives a substantial ascending influence from lower brainstem neurons that contain acetylcholine, serotonin, dopamine, noradrenaline, adrenaline, neuropeptides such as M-Enk and, to a lesser degree, substance P and CRF, and some u~own neuro~~smitte~ or neuromodulators. In future studies, we hope to determine the exact anatomical organization and physiological significance of these lower brainstem afferents to the posterior hypothalamus. ACKNOWLEDGEMENTS The authors with to thank Dr. H. Kimura for his generous gift of serotonin antisera and Dr. L. Denoroy for PNMT antisera. We also thank Dr. M. J. Carew for correction of the English. This work was supported by INSERM U 52, CNRS U.A. 1195, and DRET (Grant 87/215).

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B~INS~~

AFFERENTS

TO THE POSTERIOR