Afferent projections to the cholinergic pedunculopontine tegmental nucleus and adjacent midbrain extrapyramidal area in the albino rat. I. Retrograde tracing studies.

THE JOURNAL OF COMPARATIVE NEUROLOGY 321515-543 (1992)

Afferent Projections to the Cholinergic Pedunculopontine Tegmental Nucleus and Adjacent Midbrain Extrapyramidal Area in the Albino Rat. I. Retrograde Tracing Studies TERESA L. STEININGER, DAVID B . RYE, AND BRUCE H. WAINER Committee on Neurobiology (T.L.S., B.H.W.) and Departments of Neurology (D.B.R.) and Pharmacological and Physiological Sciences (B.H.W.), The University of Chicago, Chicago, Illinois 60637

ABSTRACT The afferent connections of the pedunculopontine tegmental nucleus (PPT) and the adjacent midbrain extrapyramidal area (MEA) were examined by retrograde tracing with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Major afferents to the PPT originate in the periaqueductal gray, central tegmental field, lateral hypothalamic area, dorsal raphe nucleus, superior colliculus, and pontine and medullary reticular fields. Other putative inputs originate in the paraventricular and preoptic hypothalamic nuclei, the zona incerta, nucleus of the solitary tract, central superior raphe nucleus, substantia innominata, posterior hypothalamic area, and thalamic parafascicular nucleus. The major afferent to the medially adjacent MEA originates in the lateral habenula, while other putative afferents include the perifornical and lateral hypothalamic area, periaqueductal gray, superior colliculus, pontine reticular formation, and dorsal raphe nucleus. MEA inputs from basal ganglia nuclei include moderate projections from the substantia nigra pars reticulata, entopeduncular nucleus, and a small projection from the globus pallidus, but not the subthalamic nucleus. Dense anterograde labeling was observed in the substantia nigra pars compacta, entopeduncular nucleus, subthalamic nucleus, globus pallidus, and caudate-putamen only following WGA-HRP injections involving the MEA. The results of this study demonstrate that the PPT and MEA share many potential afferents. Remarkable differences were found that support distinguishing between these two nuclei in future studies regarding the functional organization of the midbrain and pons. The results, for example, confirm our previous observations that the largely reciprocal connections between the midbrain and basal ganglia distinguish the MEA from the PPT. Merents from the lateral habenula and contralateral superior colliculus represent extensions of more traditional basal ganglion circuitry which further delineate the MEA from the PPT. The results are discussed with respect to the important role of the midbrain and pons in behavioral state control and locomotor mechanisms. D 1992 Wiley-Liss, Inc. Key words: mesopontine tegmentum, WGA-HRP, behavioral state control, basal ganglia

The brainstem cholinergic nuclei, the pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT), project diffusely to virtually the entire thalamus (Hallanger et al., '87) and widespread forebrain structures (Hallanger and Wainer, '881, as well as to large areas of the pontine and medullary reticular fields (Rye et al., '88); they are believed to participate in behavioral state control functions such as arousal and rapid-eye-movement (REM) sleep (see Steriade and McCarley, '90; Jones, '91 for O 1992 WILEY-LISS, INC.

review). The rat homologue of the human PPT (Jacobsohn, '09) was recently defined in our laboratory (Rye et al., '87). The PPT in the rat is a collection of large, darkly staining neurons in close association with the ascending limb of the superior cerebellar peduncle that extends from the caudal pole of the red nucleus to the rostra1 parabrachial nucleus. These PPT neurons were found to correspond entirely to Accepted March 4,1992

Abbreviations 3N 3V 4N 4V 6 IN In 10 12 ac AD A1 Amb, AMB

AP Aq AV AVPV Barr EST BST a1 BST vl BST vm Cb Ch int Cb lat Cb med cc CeA cg Cll CM CnF, Cnf CP CPU CSN CTF cu cu DBB dLGN DLL DMH DR dtb DTg ec ECu EP EW fr fx g7 Gi Giv GP Gr GTF IC ic I0 IP KF LC LD LDT LHAa LHAP LHAt LHb 11 LPO LRt LS LVe MD MEA mes 5 meV MeVn MG Mgc MHb

oculomotor nucleus third ventricle trochlear nucleus fourth ventricle abducens nucleus facial motor nucleus facial nerve dorsal motor nucleus of the vagus nerve hypoglossal nucleus anterior commissure anterodorsal thalamic nucleus agranular insular cortex nucleus ambiguus area postrema cerebral aqueduct anteroventral thalamic nucleus anteroventral paraventricular hypothalamic nucleus Barrington’s nucleus bed nucleus of the stria terminalis bed nucleus of the stria terminalis, anterolateral division bed nucleus of the stria terminalis, ventrolateral division bed nucleus of the stria terminalis, ventromedial division cerebellum intermediate cerebellar nucleus lateral cerebellar nucleus medial cerebellar nucleus corpus callosum central nucleus of the amygdala cingulate cortex commissure of the lateral lemniscus central medial thalamic nucleus cuneiform nucleus cerebral peduncle caudoputamen central superior rapbe nucleus central tegmental field cuneate nucleus cuneate fasciculus diagonal band of Broca dorsal lateral geniculate nucleus dorsal nucleus of the lateral lemniscus dorsomedial hypothalamic nucleus dorsal raphe nucleus dorsal tegmental bundle dorsal tegmental nucleus external capsule external cuneate nucleus entopeduncular nucleus Edinger-Westphal nucleus fasiculus retroflexus fornix genu of the facial nerve medullary gigantocellular field ventral gigantocellular field globus pallidus gracile nucleus gigantocellular tegmental field inferior colliculus internal capsule inferior olivary complex interpeduncular nucleus Kolliker-Fuse nucleus locus ceruleus laterodorsal thalamic nucleus laterodorsal tegmental nucleus lateral hypothalamic area, anterior lateral hypothalamic area, posterior lateral hypothalamic area, tuheral lateral habenular nucleus lateral lemniscus lateral preoptic area lateral reticular nucleus lateral septal nucleus lateral vestibular nucleus mediodorsal thalamic nucleus midbrain extrapyramidal area mesencephalic tract of the trigeminal nerve mesencephalic tract of the trigeminal nerve mesencephalic trigeminal nucleus medial geniculate nucleus magnocellular reticular formation medial habenular nucleus

ml mlf MnR Mo5 MPO MRF MS mt MVe NAc NTS ot ox PAGd PAGl PAGvl PBc PBd PBel PBexl PBg PBi PB1 PBm PBsl, PBs PBv PC PF PHA Pir PL pMoVn Pn PPT PPT-pc Pr5 PRF PrH PTA PTF PVH PVT PY Pslc Re Rh RMg RN RRF Rt RTg Sag

sc

SCP SI sm SNc SN1 SNr sol SON SP5 SP5 SPF st STh ts TZ tz VL vLGN VLL VM VMH WL WM VTA VTg xscp ZI ZId ZIV

medial lemniscus medial longitudinal fasiculus median raphe nucleus motor nucleus of 5 medial preoptic area medullary reticular formation medial septal nucleus mammilothalamic tract medial vestibular nucleus nucleus accumbens nucleus of the solitary tract optic tract optic chiasm periaqueductal gray, dorsal periaqueductal gray, lateral periaqueductal gray, ventrolateral parabrachial nucleus, central lateral subnucleus parabrachial nucleus, dorsal lateral subnucleus parabrachial nucleus, external lateral subnucleus parabrachial nucleus, extreme lateral subnucleus parabigeminal nucleus parabrachial nucleus, inferior lateral parabrachial nucleus, lateral division parabrachial nucleus, medial division parabrachial nucleus, superior lateral subnucleus parabrachial nucleus, ventral lateral subnucleus posterior commissure parafasicular thalamic nucleus posterior hypothalamic area piriform cortex paralemniscal nucleus paratrigeminal motor nucleus pontine nuclei pedunculopontine tegmental nucleus pedunculopontine tegmental nucleus, pars compacta principle sensory trigeminal nucleus pontine reticular formation nucleus prepositus hypoglossi pretectal area pontine tegmental field paraventricular nucleus of the hypothalamus paraventricular thalamic nucleus pyramidal tract pyramidal decussation reuniens thalamic nucleus rhomboid thalamic nucleus nucleus raphe magnus red nucleus retrorubral field reticular nucleus of the thalamus reticulotegmental nucleus nucleus sagulum superior colliculus superior cerebellar peduncle substantia innominata stria medullaris substantia nigra, pars compacta substantia nigra, pars lateralis substantia nigra, pars reticulata solitary tract supraoptic nucleus spinal nucleus of 5 spinal tract of trigeminal nerve subparafasicular thalamic nucleus stria terminalis subthalamic nucleus tractus solitarius nucleus of the trapezoid body trapezoid body ventrolateral thalamic nucleus ventral lateral geniculate nucleus ventral nucleus of the lateral lemniscus ventromedial thalamic nucleus ventromedial hypothalamic nucleus ventroposterior lateral thalamic nucleus ventroposterior medial thalamic nucleus ventral tegmental area ventral tegmental nucleus decussation of the superior cerebellar peduncle zona incerta zona incerta, dorsal division zona incerta, ventral division

AFFERENTS TO THE MESOPONTINE TEGMENTUM

517

the distribution of cholinergic neurons as visualized with 10% sucrose in PB at 4"C, and finally with 100 ml of 30% choline acetyltransferase ( C U T ) immunohistochemistry sucrose in PB at 4°C. The brains were removed, equili(Rye et al., '87). Previous to this analysis, the PPT was brated in 30% sucrose, and sectioned on a freezing microconsidered as a larger, ill-defined, more heterogeneous tome at a thickness of 40-50 km into four series. Sections region of the mesopontine tegmentum, which in part were collected in 0.01 M phosphate buffer, pH 7.4, containaccounted for the numerous, seemingly unrelated proposed ing 0.9% sodium chloride (PBS) at 4°C. One series of functions and connectivity ascribed to it in different re- sections was processed for the visualization of WGA-HRP ports. For example, electrophysiological studies have identi- by the tetramethylbenzidine (TMB)-HRP histochemical fied a motor pattern generator in the region of the meso- procedure of Mesulam ('82). The localization of injection pontine tegmentum (Shik et al., '66; Skinner and Garcia sites into the PPT was confirmed in selected cases by Rill, '84; Garcia Rill, '86; Garcia Rill et al., '871, which has simultaneous immunohistochemical visualization of cholinbeen ascribed to the PPT. In addition, reciprocal connec- ergic neurons and retrograde tracer. In these cases, additions between the mesopontine tegmentum and extrapyra- tional series were processed in the following manner: 1) midal motor nuclei (EPMN) have been frequently ascribed visualization of the retrograde tracer with the TMB-HRP to the PPT (Jackson and Crossman, '81a; Saper and Loewy, histochemical procedure of de Olmos et al. ('78)followed by '82; Garcia Rill, '86; Moriizumi et al., '88). Recent anatomi- the stabilization of the reaction product with a solution of cal studies in this laboratory have utilized anterograde diaminobenzidine, cobalt acetate, and hydrogen peroxide as tracing techniques (Rye et al., '87) as well as retrograde described by Rye et al. ('84a); 2) ChAT immunohistochemistracing combined with ChAT immunohistochemistry (Rye try and appropriate controls using the peroxidase-antiperoxet al., '87; Lee et al., '88) to determine that mesopontine idase method (Levey et al., '83); and 3 ) visualization of the tegmental reciprocal connectivity with the EPMN describes retrograde tracer with the stabilized TMB reaction product a region medially adjacent to the PPT for which we have in combination with ChAT immunohistochemistry (Wainer introduced the term midbrain extrapyramidal area (MEA). and Rye, '84; Rye et al., '84b). The sections were mounted The predominant connectivity distinguishingthe PPT from onto gelatin-coated slides, air dried, counterstained with adjacent structures including the MEA are its widespread neutral red, dehydrated rapidly through ethanol and xyefferent connections to "specific" thalamic nuclei (Hal- lene, and coverslipped with Histoclad mounting resin. Tissue sections were examined with a Leitz Orthoplan langer et al., '87). No comprehensive study has yet been made of specific microscope (E. Leitz Inc., Rockleigh, NJ) under brightfield afferents to the cholinergic PPT or many adjacent areas and polarized optics for the identification of anterograde recognizing these recent anatomical distinctions. Our goals and retrograde labeling. Nuclear boundaries were deterare to: 1)investigate the afferent connectivity of the PPT in mined with the aid of the rat brain atlas of Paxinos and order to understand further the circuitry in which it Watson ('82). Nuclear divisions recognized within the participates; and 2) attempt to differentiate further the mesopontine tegmentum follow the convention adopted by PPT from the MEA and other adjacent cell groups by Rye et al. ('87). In addition, subnuclear organizations addressing the specificity of these afferents. In the present recognized within the periaqueductal gray by Beitz ('85), study, we have analyzed retrograde labeling following within the medial preoptic area by Simerly and Swanson injections of wheat germ agglutinus-horseradishperoxidase ('86), and of the parabrachial nucleus by Fulwiler and Saper (WGA-HRP)into discrete areas of the mesopontine tegmen- ('84) were adopted in our identification of retrogradely tum to identify the afferents to this region. Because of the labeled neurons. configuration of the PPT nucleus, with its long rostralcaudal axis and ventral-dorsal shift, the PPT is adjacent to many different cell groups at each level. Therefore, it is RESULTS imperative to perform a detailed retrograde analysis based To investigate the afferent projections to the PPT and on a series of small, closely spaced, and overlapping injections throughout the nucleus and adjacent regions in order MEA, the retrograde tracer WGA-HRP was injected into to understand better the topography of afferents to this the region of the mesopontine tegmentum in 32 animals. region. Further studies confirming these putative afferents Given the proximity and admixture of the cell groups in this region (see Rye et al., '871, and the diffusion of tracer to will be addressed in subsequent reports in this series. adjacent regions, no injection site was limited to a single discrete cell population. Cases in which the injection sites were restricted to the PPT (cases R220, R206, R196, and MATERIALS AND METHODS R190) and MEA (cases R192, R205, and R210) with miniA total of 32 adult male Sprague-Dawley rats (250-325 g) mal spread of tracer to adjacent structures were selected for were used. The animals were anesthetized with chloral analysis. Control injections localized in the parabrachial hydrate (300 mg/kg) and placed in a Kopf stereotaxic nucleus, retrorubral field, or pontine tegmental field were apparatus (David Kopf Instruments, Torrance, CAI. Injec- also analyzed. The remaining 22 cases exhibited injections tions (3-12 nl) of either 1%or 2.5% WGA-HRP (Sigma) in that often involved more than three nuclear groups in the 0.1 M phosphate buffer were made via pressure from a glass mesopontine tegmentum and served to confirm the observamicropipette (diameter 10-30 km) with a picopuffer air tions made with more discrete tracer injections. The location of injection sites was defined by a zone of pressure system (Amaral and Price, '83). Following a 48-72 hour survival period, the animals were reanesthetized and dense and uniform deposit of TMB reaction product in the perfused transcardially over 2-4 minutes with 40-80 ml of neuropil. The injection site was surrounded by a less dense calcium-free Tyrode's buffer containing 50 units/ml hep- "halo" of TMB deposit, where the reaction product was arin, followed by perfusion with 300 ml of 3% paraformalde- observed in the neuropil, but where individual labeled hyde and 0.1-0.5% glutaraldehyde in 0.1 M phosphate axons and perikarya could be detected. It has been considbuffer (PB, pH 7.4), followed by perfusion with 100 ml of ered that the WGA-HRP tracer is not effectively taken up

518

T.L. STEININGER ET AL.

C

Figs. 1 4 . Injection sites are depicted on representative sections at four levels of the mesopontine tegmentum (A-D); derived from camera lucida drawings of reference tissue stained only for the demonstration of ChAT immunoreactivity. These figures illustrate the location of injection sites made into the pedunculopontine tegmental nucleus (cases R220 and R196) (Fig. 1) and (cases R190 and R206) (Fig. 21, the

midbrain extrapyramidal area (cases R192, R205, and R210) (Fig. 3), and surrounding mesopontine tegmentum, including the parabrachial nucleus (case R194), retrorubral field (case R189), and pontine tegmental field (case R198) (Fig. 4). Shaded areas represent the extent of WGA-HRP diffusion at the injection locus. Scale bar = 1mm.

and transported by terminals in this “halo” area (Mesulam, ’82). The appearance of anterograde labeling in PPT efferent structures (e.g., thalamic relay nuclei) (Hallanger et al., ’87; Hallanger and Wainer, ’88) and labeling of established connections of adjacent cell groups (e.g., the parabrachial nucleus) (Fulwiler and Saper, ’84) aided in defining the involvement of these cell groups within injection sites. Due to the limitations of the WGA-HRP tracing technique, such as the uptake of tracer by dendrites that extend into the injection site and the diffusion of tracer to adjacent areas, it was difficult to assess the nature of retrograde

labeling in structures immediately adjacent to the injection site.

Afferents to the PPT Four cases in which the WGA-HRP injection was restricted to the region of the PPT were selected for analysis. The extent of the injection sites in these cases is depicted in Figures 1and 2. Analysis of labeling is presented in Table 2. Case R220. The injection site in case R220 (Figs. 1and 5 ) encompasses much of the region of the PPT neurons,


519

Figure 2

with little diffusion of tracer into adjacent structures. The WGA-HRP injection in this case was made with the injection pipette angled (45") in a caudal approach. This approach allowed a greater rostrocaudal diffusion of tracer, with limited diffusion in the dorsoventral direction. The injection site was centered in the pars compacta at a level just caudal to the decussation of the superior cerebellar peduncle, and extended rostrally to the level of the oculomotor nucleus. At its caudal extent, the injection site involved the rostral parabrachial nucleus around the micropipette tract, and the tracer slightly spread to the medially adjacent MEA. This case was utilized as an example to illustrate the retrograde labeling resulting from injections of tracer into the PPT. Retrograde labeling in this case is illustrated in Figures 6 and 7 and analysis of labeling is presented in Tables 1and 2.

Telencephalon Bed nucleus of the stria terminalis (BST). A moderate density of labeling was observed in the BST, mainly in the ventrolateral and preoptic subdivisions, with fewer neurons seen in the ventromedial and anterior lateral subnuclei (Fig. 6C). Central nucleus of the amygdala (CeA). A small number of labeled neurons was seen in the medial division of CeA (Fig. 6D). Labeling was not observed in other amygdaloid nuclei. Substantia Znnominata (SI). Labeling in the SI was observed in two regions. A few labeled neurons were observed in a region of the rostral SI, ventral to the anterior commissure (Fig. 6B,C), and caudally, several labeled neurons were observed in a region of SI that is encapsulated by fibers of the internal capsule, medial to the optic tract, in


520 TABLE 1. Number of Labeled Neurons Following Injections Into the PPT or MEA'

TABLE 2. Analysis of Retrograde Labeling Following Injections Into the PPT'

Case no

Telencephalon Profrontal cortex Septumidiagonal hand Bed nucleus of the stria terminalis Central nucleus of the amygdala Substantia innominata Diencephalon Medial preoptic area Lateral preoptic area Paraventricular nucleus Lateral hypothalamic area, anterior Lateral hypothalamic area, tuheral Lateral hypothalamic area, posterior Posterior hypothalamic area Perifornical area Lateral habenula Entopeduncular nucleus Zona incerta Mesencephalon Superior colliculus Substantia nigra, pars compacta Suhstantia nigra, pars reticulata Suhstantia nigra, pars lateralis Central tegmental field Ventral tegmental area Retrorubral field Edinger-Westphal nucleus Central superior raphe Periaqueductal gray, dorsal Periaqueductal gray, lateral Periaqueductal gray, ventrolateral Pons Dorsal raphe nucleus Median raphe nucleus Cuneiform nucleus Laterodorsal tegmental nucleus Parabrachial nucleus, lateral Parabrachial nucleus, medial Locus ceruleus Barringon's nucleus Pontine tegmental field Gigantocellular tegmental field Cerebellar nucleus, lateral Cerebellar nucleus, interposed Medulla Nucleus prepositus hypoglossi Spinal sensory trigeminal nucleus Cuneate nucleus Nucleus of the solitary tract, rostral Nucleus of the solitary tract, caudal Gigantocellular reticular field Ventrolateral medulla

Case no

R220

R192

1112 411 5710 1810 101 1

210 010 011 010 1216

311 2817 3310 2710 1221 14 211114 6111

1qo

2014 010 9315 133 157

1515 4212 3613 519* I 3 0 1615 124* 130 10 53 202 121 811 173 501 161 206 0 5 1 113 5119 '11 '11 4714 1014 96 146 70 128 12011 190 10

$0 017 3412 68110 73 155 38118

010 011 410 261 15 84 128 193 I94

1519 2116 400 I 133 1513 15/10

23* 1174 16*12 6219 010 143* 155 53:/4 110 20 43 911 69* 116 152' 128 218 33

61119 1141 115 33 118 13 128 1111 32\18 29/21 3810 710 419 210 015 01 1 210

161 12 010

'Number of retrogradely labeled neurons (ipsilateral I contralateral! a t identified brain regions following injections of WGA-HRP into the pedunculopontine tegmental nucleus (case R220! and midbrain extrapyramidal area (case R192). Cell counts were made of every fourth 50 pm section through the brain. *Regions that were in proximity to the injection site, where labeling could not he discerned due to the presence of reaction product in the neuropil, or the presence of dense anterograde labeling.

the region of cholinergic neurons of the nucleus basalis of Meynert (NBM) (Fig. 6D,E). Other labeled regions. In the prefrontal cortex, a small number of pyramid-shaped neurons was observed in the rostral cingulate and paralimbic cortices (6A). An additional small cluster of labeled neurons was seen in the Labeling observed in rostral agranular insular cortex (AI). A1 is likely the result of parabrachial involvement in the injection site, as a dense projection from this region to the parabrachial nucleus has been demonstrated with both retrograde and anterograde tracing techniques (Moga et al., 'go), and this region remained unlabeled following injections into other regions of the PPT. A few labeled neurons were observed in the diagonal band region (Fig. 6B). Diencephalon Hypothalamus. The hypothalamus was found to be a major source of retrograde labeling. The greatest hypotha-

R220 Telencephalon Prefrontal cortex Septumidiagonal hand Bed nucleus of the stria terminalis Central nucleus of the amygdala Substantia innominata Diencephalon Lateral preoptic area Paraventricular nucleus Dorsomedial nucleus Lateral hypothalamic area, anterior Lateral hypothalamic area, tuberal Lateral hypothalamic area, posterior Posterior hypothalamic area Perifornical area Lateral hahenula Zona incerta Mesencephalon Superior colliculus Suhstantia nigra, pars compacta Substantia nigra, pars reticulata Substantia nigra, pars lateralis Central tegmental field Ventral tegmental area Retrorubral field Edinger-Westphal nucleus Central superior raphe Periaqueductal gray, dorsal Periaqueductal gray, lateral Periaqueductal gray, ventrolateral Pons Dorsal raphe nucleus Cuneiform nucleus Laterodorsal tegmental nucleus Parabrachial nucleus, lateral Parabrachial nucleus, medial Locus ceruleus Barrington's nucleus Nucleus raphe magnus Pontine tegmental field Gigantocellular tegmental field Cerebellar nucleus, lateral Cerhellar nucleus, interposed Medulla Nucleus prepositus hypoglossl Nucleus of the solitary tract, rostral Nucleus of the solitary tract, caudal Spinal sensory trigeminal nucleus Cuneate nucleus Medullary gigantocellular field Ventrolateral medulla

R206

-I+I-

R196

R190

++I-

nd

+I-

nd

+/-

++I-

+++

+I-

+I+I-

+I+I-

+I++/+

+I+I-

+I+/++I-

++/-

+I-

+I-

++]+

+I-

+++I+

+++

+I+

++/-

++++I+

++/-

-I-

++I-

+++

-I-I-

-I-I++I+

+I+

+++/+

-I-I+I+++I+

+I-

-I-

-I-

-/-

+*/-

+I+I-

++/+++I++ ++I-

++[++I++++I+ +I-

+++*/+ + ++ +++/+

+++

+/-

+I+++I+ +I++*/+

++++I++

+++

++++I+ +I-

+I*I++

++*I+

-

-

+

+

+I-

+++ / + t+++/++

+++I+

++++ +*/-

++I+

++I-

++I-

++/-

*I-

++*]-

++*I+

*I-

+*I-I+I+ ++I+

+*I+

++I++

+

++I+

++I+

+++I-

++I+

++++ +/-

+*I-

+

++++I++

+/-

+ / -

++I+I-

t+++]+

++++ ++I+

+ / -

++I+

-

++++

++++I++

++I+I-

+I-

+++

+I-

+++

+/-

+ I -t++l+t+

+++I++

++It++l++

t+++l+

++ *I++I+ ++I+I-

-+ +I-

-

++I++ ++I+

+++ 1 +

+I+

++I-

-I-

+I+

+I+

-I-

-I-

+I+ ++I-

-I-

-/+

-I-

+I++I+

+++

-I+

-I-

++I+ +I+

++/+ ++I+

++I+ +I+

-I-

++/+ -/-

'Semiquantitative analysis of retrograde labeling following injections centered in the pedunculopontine tegmental nucleus. Case numbers refer to the injections described in Figures 1and 2 and in the text Retrograde labeling in brain regions in this Table and in Tables 3 and 4 is indicated as follows: Labeling is presented semiquantitatively (ipsilateral 1 contralateral!, which reflects the relative density of labeled neurons per region, in one series, expressed on a scale of 0 to 4: - , O-9 labeled neurons; +, 10-40; + +, 41-100; +++, 101-200; ++++, morethan200neurons. *Regions that were in proximity to the injection site, and could not he analyzed. nd, laheling was not determined.


521

B CTf

D

R192 Figure 3

lamic contribution to retrograde labeling was the lateral hypothalamic area (LHA). The LHA was roughly divided into three rostral-caudal regions as defined by Saper et al., (’79). The anterior region (LHAa) extended roughly from the level of the rostra1 paraventricular hypothalamic nucleus (PVH) to the ventromedial nucleus (VMH). The tuberal region (LHAt) extended from the level of the VMH to the subthalamic nucleus (STh). The posterior region (LHAp) extended from the level of STh to the mammillary recess. The posterior hypothalamic area (PHA) extended from the LHAp to the level of the mesencephalon. Dense retrograde labeling was observed in the tuberal and posterior LHA, with only a moderate density observed in the anterior region. The population of labeled neurons was scattered diffusely throughout the LHA, although the

neurons were somewhat concentrated at the lateral aspect of LHA, adjacent to the internal capsule (Fig. 6E,F). Farther caudally, labeled neurons describe a “cap” around the medial edge of the cerebral peduncle, adjacent to the subthalamic nucleus. A moderate density of labeled neurons was observed in the PVH. These labeled neurons were located primarily in the parvicellular subdivision of the nucleus (Fig. 6D). In the anterior hypothalamus, a moderate number of retrogradely labeled neurons was seen scattered throughout the lateral preoptic area (LPO) (Fig. 6C). Thalamus. Retrograde labeling in the thalamus was seen only at posterior levels (Fig. 6G). A few retrogradely labeled neurons ( < 5 ) were observed in the ventral lateral geniculate nucleus and the reticular nucleus. A few


522

B

Figure 4

labeled neurons were observed in the subparafasicular nucleus, and a moderate number of labeled neurons was seen in the parafasicular nucleus; they were located dorsal to the fasciculus retroflexus. Diffuse anterograde labeling was seen in nearly all thalamic nuclei, particularly the lateral geniculate and reticular nuclei; this observation verifies our previous identification of PPT efferents to the thalamus employing retrograde (Hallanger et al., '87) and anterograde (Hallanger and Wainer, '88) methodologies. Other labeled regions. A moderate density of labeled neurons was observed in the zona incerta (ZI). Labeled neurons were more numerous at rostra1 levels (Fig. 6F), and at caudal levels, the labeled neurons were seen mainly in the ventral subdivision (Fig. 6G). In addition, a few labeled neurons were observed bilaterally in the lateral

habenula (Fig. 10B). No labeling was observed in the entopeduncular nucleus (Fig. 10D). Mesencephalon. Retrograde labeling in the mesencephalon represented a large proportion of the total labeling observed. The greatest retrograde labeling was observed in the central tegmental field (CTF), the periaqueductal gray (PAG), and the dorsal raphe nucleus (DR). Central tegmental field. Retrograde labeling in the CTF was very dense and was considered a major contributor to total labeling. The CTF occupies a major portion of the mesencephalic tegmentum and extends from the mesodiencephalic junction to the cuneiform nucleus in the mesopontine tegmentum. The CTF is bordered dorsally by the superior and inferior colliculi, medially by the periaqueductal gray, laterally by the medial geniculate nucleus and the

AFFERENTS TO THE MESOPONTINE TEGMENTUM brachium of the inferior colliculus (BIC), and ventrally by the red nucleus, retrorubral field, and MEA. Labeled neurons were found diffusely scattered throughout the region (Figs. 6H, 7A,B). Labeling appeared evenly distributed along the rostral-caudal axis, although at caudal levels neuronal labeling was partially obscured by the reaction product in the vicinity of the injection site, as well as by the presence of anterogradely labeled fibers coursing rostrally from the injection site. Periaqueductal gray. Dense retrograde labeling was observed in the PAG. The PAG was subdivided into quadrants as defined by Beitz ('85) on the basis of anatomical connections and cytoarchitecture. The lateral quadrant was the most heavily labeled, and labeled neurons were concentrated in its ventral portion (Fig. 7A-C). The ventrolateral quadrant was also densely labeled. The dorsal quadrant of PAG contained a low to moderate density of labeled neurons (Figs. 6H, 7A,B). Dorsal raphe nucleus. Dense retrograde labeling was observed in DR. Labeled neurons were closely packed and were concentrated in the dorsal region of the nucleus. Dense labeling was observed at rostral to middle levels of the nucleus (Fig. 7C), and was much less dense at caudal regions of the nucleus (Fig. 7D). Only a moderate input from the central superior raphe nucleus (CSN) was observed. Superior colliculus. Moderately dense retrograde labeling was observed throughout the deep layers of the superior colliculus (SC) (Fig. 7A-C). On the contralateral side, a small number of labeled neurons was concentrated at the ventrolateral portion of the SC, dorsal to the brachium of the inferior colliculus. Other labeled regions. A small to moderate number of retrogradely labeled neurons was observed in the substantia nigra pars compacta (SNc), pars reticulata (SNr) and pars lateralis (SNl) (Figs. 6H, 7A,B). Retrogradely labeled neurons were most frequently observed in the pars lateralis, which is situated dorsolateral to the SNc and SNr and medial to the lateral aspect of the cerebral peduncle. Labeled neurons in the pars reticulata were found mainly at lateral aspects of the subnucleus (Fig. 6H). A few labeled neurons were observed in the pars compacta. Anterograde labeling was seen as a bundle of labeled fibers dorsal to the pars compacta, with only sparse anterograde labeling observed over the compacta neurons (Fig. 10F). Retrograde labeling in the retrorubral field (RRF) was moderately dense where retrograde labeling was partially obscured by reaction product in the vicinity of the injection site, as well as by fibers from the injection site that coursed rostrally through the tegmentum. A small number of labeled neurons was observed in the cuneiform nucleus and the contralateral MEA. In the contralateral PPT, a small number of large neurons (16-20 Fm) was observed. The cholinergic identity of these neurons was verified on adjacent tissue processed for the simultaneous visualization of retrogradely transported WGA-HRP and ChAT immunohistochemistry. Sparsely labeled regions included the ventral tegmental area and the Edinger-Westphal nucleus. Pons Pontine reticular formation. Moderately dense retrograde labeling was observed in the pontine reticular formation (PRF). The rostral portion of the pontine reticular formation is the pontine tegmental field (PTF), which is also termed the nucleus pontis oralis. The PTF was differentiated from the parvicellular midbrain CTF by the presence

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Fig. 5. Typical WGA-HRP injection, in this case located in the pedunculopontine nucleus, as visualized with tetramethylbenzidine, viewed under brightfield optics. Scale bar = 400 ym.

of scattered medium-sized and very few large-sized neurons. At caudal levels of the pons, many giant-sized cells are present. This region of PRF is termed the gigantocellular tegmental field (GTF), which is also known as the nucleus pontis caudalis. Retrogradely labeled neurons were more numerous at rostral levels (PTF) than at caudal levels (GTF) and appeared to be concentrated medially (Fig. 7E,F). Numerous anterogradely labeled fibers were observed descending from the injection site in the lateral aspect of PRF. Cerebellum. The deep cerebellar nuclei contained a large number of retrogradely labeled neurons, which were found mainly in the lateral (dentate) and interposed (interpositus) nuclei. No significant labeling was observed in the medial (fastigial) nucleus. Retrograde labeling seen in the deep cerebellar nuclei was likely due to the uptake of tracer by axons ascending in the superior cerebellar peduncle, which were damaged by the injection micropipette. Labeling of the deep cerebellar nuclei was observed in a previous study in which WGA-HRP injections were made into the parabrachial nucleus, which surrounds the superior cerebellar peduncle caudal to the PPT (Moga et al., '90). A dense projection from the cerebellar nuclei to the PPT or PB is unlikely, in that no evidence of anterograde labeling was observed in the PB or in the PPT following injections of


524

A

Figs. 6 and 7. Line drawings of representative sections (arranged rostra1 to caudal; A-H and A-K, Figs. 6 and 7, respectively) made with the aid of a camera lucida, demonstrating the distribution of retrograde labeling following an injection of WGA-HRP into the pedunculopontine tegmental nucleus (case R220). Each dot represents two retrogradely labeled neurons in a single 50 p m thick section. Scale bar in Figure 6 = 4 mm and applies to Figure 7 as well.

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526 tracer in the dentate or interpositus nuclei (Moga et al., ’90; personal observation). Laterodorsal tegmental nucleus (LDT). A moderate number of labeled neurons was seen in the LDT. Retrogradely labeled neurons were medium to large in size (18-23 pm), and these neurons were double labeled in adjacent sections processed additionally for ChAT immunohistochemistry. A few smaller noncholinergic neurons (8-10 pm) were also labeled. Parabrachial nucleus (PB). The injection site in this case involved the rostral portion of PB, which is situated immediately adjacent to the caudal PPT. Farther caudally, the reaction product in the neuropil of PB was much less dense than at the locus of the injection, and several neurons with dense reaction product in their soma could be distinguished from neuropil reaction product. Numerous retrogradely labeled neurons were seen in the superior lateral, dorsal lateral, and medial subnuclei, and also the waist region. In addition, labeled fibers were seen descending from the injection site through the region of PB and reticular formation ventral to PB. A few labeled neurons were observed contralateral to the injection site. Other labeled regions. Sparsely labeled regions included the locus ceruleus (LC),pontine central gray, Barrington’s nucleus, and the nucleus raphe magnus; only a few labeled neurons were observed in each of these regions. Labeled neurons observed in the pontine central gray were located ventral to the rostral neurons of LC. Medulla Medullary reticular formation (MRF). Overall, labeling in the medullary reticular formation was less dense than labeling observed in PRF. Labeled neurons were scattered bilaterally throughout the gigantocellular region, and appeared more concentrated in medial regions (Fig. 7G-K). In addition, a cluster of large (15-35 pm) neurons was labeled in the contralateral dorsomedial reticular formation, ventral to the nucleus prepositus hypoglossi (Fig. 7H). This MRF region in the cat was previously found to have projections to the vestibular nuclei, the nucleus prepositus, and the abducens nucleus (Graybiel, ’77). Rostra1 ventrolateral medulla (RVL). A small number of labeled neurons was observed in RVL (Fig. 71,J). Nucleus of the solitary tract (NTS). A moderate number of retrogradely labeled neurons was observed in the NTS. Labeled neurons were concentrated in the region of the nucleus caudal to the obex, and extended caudally to the rostral spinal cord (Fig. 7J,K) (i.e., the commissural NTS), and closely approximated the region occupied by the A2 cell group of Dahlstrom and Fuxe (’64).Fewer labeled neurons were observed in more rostral regions of the NTS (Fig. 71). Other labeled regions. Sparsely labeled regions included the lateral vestibular nucleus and the contralateral spinal trigeminal nucleus. Only a few retrogradely labeled neurons were observed in the nucleus prepositus hypoglossi (PrH) (Fig. 7H). Spinal cord. The spinal cord of this case was dissected into segments (cervical, thoracic, etc) with the aid of a dissection microscope, and frozen-sectioned in the horizontal plane. The examination of these horizontal sections revealed moderate anterograde labeling in laminae 1-111, as well as several retrogradely labeled neurons, primarily encountered at cervical levels. Labeled neurons were also seen farther ventrally, in laminae V-VII. No labeled neurons were observed in the ventral horn. The thoracic and

sacral levels were relatively unlabeled; however, a few labeled neurons were observed in the lumbar spinal cord.

Other PPT injections Case R206. The WGA-HRP injection site in this case was centered in the mid level PPT, rostral to the injection site center of R220 (Fig. 2). The injection in case R206 was smaller than that in R220, which accounted for the reduction in total labeling observed. There was a slight diffusion of tracer to the rostral MEA, located dorsomedial to the PPT at this level, which appeared to be less extensive than the MEA involvement in case R220. At its caudal extent, the injection site was ventral to that of R220, and surrounded the medial and ventral aspect of the ascending superior cerebellar peduncle. The injection site did not involve the rostral PPT or retrorubral field, and also did not extend as far caudal as to involve the parabrachial nucleus. Anterogradely labeled fibers were more apparent in this case when compared to case R220. The reason for this was unknown, but may be due to a subtle difference in the fixation of the tissue, or in the histochemical procedure. Anterograde labeling was observed in the region of the nucleus basalis of Meynert (NBM) and in the reticular thalamic nucleus (Rt), which are known targets of PPT innervation (Hallanger et al., ’87; Hallanger and Wainer, ’88).

The pattern of retrograde labeling in this case was similar to that observed in R220 (Table 2). The densest retrograde labeling was observed in the periaqueductal gray, the dorsal raphe nucleus, and the central tegmental field. Moderate labeling was observed in the tuberal and posterior LHA, zona incerta, pontine reticular formation, medullary gigantocellular field, and nucleus of the solitary tract. In contrast to the labeling in case R220, the rostral insular cortex remained unlabeled. No labeling was observed in the lateral habenula, and very little labeling was observed in the substantia nigra. Structures known to project strongly to the parabrachial nucleus were labeled less well or not at all when compared to case R220. For example, less labeling was observed in the posterior lateral hypothalamic area, bed nucleus of the stria terminalis, and central nucleus of the amygdala. The “cap” of labeling in the posterior LHA was also not observed, and labeling was absent in the Edinger-Westphal nucleus and in the rostral precommissural portion of the nucleus of the solitary tract. In addition, there was a pronounced shift in the pattern of labeled neurons in the periaqueductal gray when compared to case R220. In case R206, labeling in the dorsal quadrant was more dense and labeling in the lateral quadrant was less dense, and was not concentrated ventrally, as in R220. Substantially less labeling was observed in the superior colliculus. Retrograde labeling was much less dense in the deep cerebellar nuclei. Case R196. The injection site in this case was centered in the rostral to midlevel of the PPT (Fig. I). The injection site involved the retrorubral field at its rostral extent, and part of the paralemniscal nucleus at its lateral edge. The injection site extended caudally to involve the pars compacts of the PPT partially. There appeared to be no involvement of the MEA or PB nucleus in the injection site. Analysis of labeling is presented in Table 2. Dense retrograde labeling was again observed in the central tegmental field, periaqueductal gray, dorsal raphe, superior colliculus, and pontine reticular formation. Moder-

527

AFFERENTS TO THE MESOPONTINE TEGMENTUM ate labeling was observed in the hypothalamus, including the lateral preoptic area, paraventricular nucleus, anterior and tuberal lateral hypothalamic area, and also in the bed nucleus, substantia innominata, zona incerta, substantia nigra pars lateralis, and caudal nucleus of the solitary tract. Although much of the labeling was similar to that observed in case R220, several differences were noted. There appeared to be a shift in the lateral hypothalamic labeling, such that the anterior region was more heavily labeled, with less labeling observed in the tuberal region, and very few labeled neurons seen in the posterior region. Several forebrain regions were more heavily labeled including the substantia innominata, lateral preoptic area, paraventricular nucleus and prefrontal cingulate cortex. No retrograde labeling was observed in the thalamus, including the parafascicular nucleus, reticular nucleus, or ventral lateral geniculate. No retrograde labeling was observed in the lateral habenula, or in the rostral insular cortex. In the brainstem, pontine reticular fields were more heavily labeled, particularly contralateral to the injection. A small number of retrogradely labeled neurons was observed in the thalamic subparafascicular nucleus, and in the pontine central gray dorsolateral to the genu of the facial nerve. Retrograde labeling in these structures was not observed in the other PPT cases presented; however, these regions were found to be consistently labeled following larger tracer injections that involved the paralemniscal nucleus, lateral to the PPT. Case R190. The injection site in this case was centered in the rostral third of the PPT, and is located medial to the injection site in case R196 (Fig. 2). The injection site involved the retrorubral field rostrally and the central tegmental field dorsally, and slightly involved the MEA caudally. Overall labeling was less dense than in the previous cases. The pattern of retrograde labeling was different from R220 as follows (Table 2). Dense retrograde labeling was observed in the central tegmental field, periaqueductal gray, and superior colliculus. Moderate labeling was observed in the pontine reticular formation, dorsal raphe nucleus, and lateral hypothalamic area. Lateral hypothalamic labeling was similar to that observed in case R220, in that labeling was most dense at tuberal and posterior levels; however, overall labeling was less dense. More retrograde labeling was observed in several forebrain regions, including the medial septalidiagonal band region, central nucleus of the amygdala, substantia innominata, and hypothalamic paraventricular nucleus. No labeling was observed in the Edinger-Westphal nucleus, or in the substantia nigra pars compacta. In the brainstem, labeling in the dorsal raphe nucleus was lighter, but substantial. In the cerebellar nuclei, the lateral nucleus was lightly labeled, and the interposed and medial nuclei were unlabeled. In this case, the injection site was far rostral to the parabrachial nucleus, and therefore retrograde parabrachial labeling could be best assessed. Labeling in PB was moderate, and observed mainly in the ventrolateral and dorsolateral subnuclei, the waist area, and to a lesser extent the medial subnucleus. Very little labeling was observed on the contralateral side; however, a few labeled neurons were observed in the contralateral superior lateral subnucleus. No labeling was observed in the nucleus of the solitary tract, and very little labeling was observed in the medullary reticular formation. Additional, substantial anterograde labeling was observed in the ventrolateral funiculus of the cervical spinal cord.

Afferents to the MEA Case R192. Case R192 is an example in which the WGA-HRP injection site was centered in the MEA and involved little of the surrounding cell groups (Fig. 3). This case will be utilized to illustrate the pattern of retrograde labeling from the MEA (Figs. 8,9). Telencephalon Substantia innominata. A moderate projection to the MEA from the substantia innominata was seen. Labeled neurons were observed ventral to the globus pallidus, just caudal to the crossing of the anterior commissure. Labeling in this region was significantly more dense than that observed in PPT injections. Caudateputamen (CPU)and globus pallidus (GP). Only occasional retrogradely labeled neurons were observed in the CPU, and a small number of labeled neurons were observed in GP (Fig. 8C). In both CPU and GP, moderate levels of anterograde labeling were present, and were particularly evident in the lateral aspect of each structure. Entopeduncular nucleus (EP). Several retrogradely labeled neurons were observed bilaterally in EP, with fewer observed contralateral to the injection (Fig. 8D, IOC). Moderate anterograde labeling in EP was also observed (Fig. lOC). Other labeled regions. No labeling was observed in the cerebral cortex, or in the septalidiagonal band complex. A few labeled neurons were seen in the contralateral ventral region of the bed nucleus (Fig. 8B). Diencephalon Epithalamus. The highest density of retrograde labeling in this case was observed in the lateral habenular nucleus (LHb) (Figs. 8E, 10A). Labeled neurons were densely clustered in the lateral division of LHb, and were heavily filled with reaction product (Fig. lOA), which was in striking contrast to the lateral habenular labeling observed in case R220 (Fig. 10B). Labeled neurons were observed bilaterally, with an ipsilateral dominance. Hypothalamus. In the hypothalamus, relatively dense labeling was seen in the LHA, where labeled neurons were located mainly at tuberal and posterior levels (Fig. 8D,E). In contrast to the pattern of labeling observed following PPT injections, labeled neurons were seen to occupy a more medial position in LHA and were numerous in the perifornical region. Caudal to the subthalamic nucleus, a few labeled neurons were seen in the lateral region of the posterior hypothalamic area. No significant labeling was observed in the preoptic region. A few labeled neurons were seen in LPO (Fig. 8C). Moderate labeling was observed in the dorsomedial nucleus, and a few labeled neurons were observed in the dorsal hypothalamic region medial to the mammillothalamic tract (Fig. 8E). Subthalamus. No labeled neurons were observed in the subthalamic nucleus (STh); however, moderate levels of anterogradely labeled fibers were observed in this nucleus. Other labeled regions. In the thalamus, retrograde labeling was seen only at caudal regions. Moderate labeling of neurons was observed in the parafascicular nucleus surrounding the fasciculus retroflexus. A few labeled neurons were observed in the subparafascicular nucleus and in the periventricular zone (Fig. 8F). A few labeled neurons were observed in the rostral zona incerta (Fig. 8D). Mesencephalon Periaqueductal gray. Dense retrograde labeling was observed in the periaqueductal gray; however, labeling was less dense than that observed in PPT injections. No signifi-

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T.L.STEININGER ET AL.

Figs. 8 and 9. The distribution of retrogradely labeled neurons in forebrain and brainstem following an injection of WGA-HRP into the midbrain extrapyramidal area (case R192) is illustrated in this series of camera lucida drawings (A-HI. Each dot represents a single retrogradely labeled neuron in a single 50 pm thick section. Scale is same as Figures 6 and 7.


529

F

G

Figure 9

530 cant labeling was seen at rostral levels (Fig. 8G). Further caudally, labeling was moderate in the lateral quadrant, where it was concentrated in a ventral position (Fig. 9A). Labeling in the ventrolateral quadrant was relatively dense. There was a slight leakage of tracer from the injection tract at the lateral edge of the lateral and ventrolateral quadrants, with a few labeled neurons observed in the vicinity of this tracer deposit (Fig. 9A). Therefore, some of the retrograde labeling observed in PAG may reflect local PAG interconnections rather than putative MEA inputs. Dorsal raphe nucleus. The density of retrograde labeling in the dorsal raphe from the MEA was comparable to that seen following PPT injections. Labeled neurons were concentrated in the dorsal region of the nucleus (Fig. 9A). Many labeled neurons were also observed in the central superior raphe, where labeling was located mainly at caudal levels of the nucleus (Fig. 8H). Substantia nigra. A dense accumulation of anterograde labeling seen over the pars compacta of the SN, which was confined to its lateral two-thirds of the compacta, served to obscure detection of retrograde labeling (Fig. 10E). Nevertheless, a small number of retrogradely labeled neurons was discernible. In the pars reticulata, labeled neurons were more numerous and were found mainly at caudal levels. No labeling was observed in the pars lateralis. Retrorubral field. Several retrogradely labeled neurons were observed in this region. However, due to the proximity of the injection site, diffuse reaction product in the neuropil and labeled fibers traveling through the region obscured a meaningful analysis of retrograde labeling. A few labeled neurons were observed on the contralateral side. Superior colliculus. In this case, leakage of tracer along the pipet track in the SC resulted in a small depost of reaction product and the labeling of neurons in the vicinity. Outside of this injection track, few labeled neurons were observed ipsilaterally. In contradistinction, contralateral retrograde labeling was robust and described a population of large neurons that was situated primarily in deep layers and concentrated in a ventrolateral position within the colliculus (Fig. 8H). Central tegmental field. Moderate levels of labeling were seen in the CTF. Labeled neurons were observed to be located rather diffusely throughout this region (Fig. 8G,H). Ventral tegmental area. Moderate retrograde labeling was seen in VTA. At caudal levels, labeled fibers emanating from the injection site traveled through this region. Other labeled regions. In the region of the PPT, a few labeled neurons were observed on the contralateral side (Fig. 9A). These neurons were small in size (6-8 pm) and triangular in appearance, suggesting that the labeled neurons were the noncholinergic neurons that are found admixed with the cholinergic PPT neurons (Rye et al., '87). ChAT immunohistochemistry was not performed in this case. The contralateral MEA contained several labeled neurons, which were observed embedded within the fibers of the superior cerebellar peduncle. A few labeled neurons were seen in the cuneiform nucleus and paralemniscal nucleus, ipsilateral to the injection site. No labeling was observed in either the dorsal or ventral nuclei of the lateral lemniscus. Several labeled neurons were seen in the EdingerWestphal nucleus (Fig. 8H). Pons Pontine reticular formation. Overall labeling in the PRF was less dense than that observed in PPT injections. A moderate number of labeled neurons was observed bilater-

T.L. STEININGER ET AL. ally in PTF. Labeled neurons on the ipsilateral side were medium to large (9-20 km), whereas only small labeled neurons (8-10 pm) were seen on the contralateral side. Caudally, the GTF was less densely labeled (Fig. 9C). Parabrachial nucleus. A relatively high density of retrograde labeling was observed bilaterally in PB. Labeled neurons were found in the dorsal lateral and central lateral subnuclei. The superior lateral and external lateral subnuclei were devoid of labeling. In the medial subnucleus, labeled neurons were moderately dense at rostral levels and absent at caudal levels. Median raphe. Relatively dense labeling was seen in the MnR that was concentrated laterally. The MnR was medial to, but not involved in, the injection site in the MEA. However, due to the proximity of this structure to the injection, no conclusions can be drawn about MnR projections to MEA. Laterodorsal tegmental nucleus. Retrograde labeling in the LDT was moderate. Labeled neurons were concentrated at rostral levels of the nucleus, where they were nearly exclusively large in size (15-20 km) (Fig. 9B,C). Caudally, a few smaller neurons were labeled. Other labeled regions. Light to moderate labeling was observed in LC. Labeled neurons were also observed rostrally in the pontine central gray, ventral to the most rostral neurons of LC. A few neurons were observed in Barrington's nucleus on the ipsilateral side. In the deep cerebellar nuclei, moderate labeling was seen in the lateral nucleus, and a few labeled neurons were seen in the interposed nucleus (Fig. 9E). The medial nucleus was devoid of labeling. Medulla. Labeling was sparse thorughout the medulla. Very little retrograde labeling was seen in the medullary reticular formation. A few labeled neurons were observed in the dorsal gigantocellular field and in the nucleus prepositus hypoglossi (Fig. 9E). In the dorsal columns, no labeling was seen in the gracile nucleus; however, a few labeled neurons were observed in the cuneate nucleus. A few retrogradely labeled neurons were observed in the dorsal cervical spinal cord. Other spinal cord afferents were not analyzed for this case.

Other MEA injections Case R205. The injection site in this case was larger than that in case R192 and was centered in the MEA at the level of the PPT pars compacta (Fig. 3). At rostral levels, the injection site involved the caudal part of the retrorubral field. At middle levels, the injection involved the dorsomedial part of PPT, and at caudal levels, the injection site included the tegmentum ventrolateral to the edge of the periaqueductal gray. There appeared to be no involvement of the parabrachial nucleus in this injection. Retrograde labeling in the ventrolateral PAG, cuneiform nucleus, and LDT was obscured by reaction product in proximity to the injection site. Anterograde labeling was observed in the striatum, globus pallidus, subthalamic nucleus, and substantia nigra pars compacta. Sparse anterograde labeling was also observed in the reticular, lateral geniculate, and ventroposterior nuclei of the thalamus, consistent with involvement of the PPT in the injection site. Analysis of labeling is presented in Table 3. Dense retrograde labeling was observed in the lateral habenula, central tegmental field, superior colliculus, dorsal raphe nucleus, and lateral hypothalamic area. Moderate labeling was observed in the substantia innominata, hypothalamic paraventricular nucleus, thalamic parafascicular

AFFERENTS TO THE MESOPONTINE TEGMENTUM TABLE 3. Analysis of Retrograde Labeling Following Injections Into the MEA' Case no. R192 Telencephalon Septum/diagonal band Bed nucleus of the stria termina-

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R210

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Central nucleus of the amygdala Striatum Globus pallidus Substantia innominata Diencephalon Lateral preoptie area Paraventricular nucleus Lateral hypothdamic area, anterior Lateral hypothalamic area, tuberal Lateral hypothalamic area, posterior Posterior hypothalamic area Perifornical area Lateral habenula Entopeduncular nucleus Zona incerta Mesencephalon Superior colliculus Substantia nigra, pars compacta Substantia nigra, pars reticulata Substantia nigra, pars lateralis Central tegmental field Ventral tegmental area Retrorubral field Edinger-Westphal nucleus Central superior raphe Periaqueductal gray, lateral Periaqueductal gray, ventrolateral Median raphe nucleus Pons Dorsal raphe nucleus Cuneiform nucleus Laterodorvdl tegmental nucleus Parabrachial nucleus, lateral Parabrachial nucleus, medial Locus ceruleus Barrington's nucleus Pontine tegmental field Gigantocellular tegmental field Cerebellar nucleus, lateral Medulla Medullary gigantocellular field Ventrolateral medulla

R205

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'Semiquantitative analysis of retrograde labeling in brain regions following injections centered in the midbrain extrapyramidal area. Case numbers refer to the injections described in Figure 3 and in the text. Retrograde labeling is indicated as in Table 2 .

nucleus, and periaqueductal gray. Several differences in labeling from R192 were noted. Forebrain regions that were more densely labeled include the bed nucleus, central nucleus of the amygdala, substantia innominata, and hypothalamic paraventricular nucleus. In the bed nucleus, labeled neurons were seen in the dorsolateral and preoptic subnuclei. Labeled neurons in SI were observed caudally, along the lateral aspect of the internal capsule, in addition to ventral pallidal labeling. In the hypothalamus, labeling in the posterior LHA resembled that of the PPT injection case R220, in that labeled neurons were concentrated along the medial border of the internal capsule. Moderate labeling was observed in the lateral habenula, which was localized to the lateral division. Moderate retrograde labeling of neurons in the thalamic parafascicular nucleus was observed. Retrograde labeling in the superior colliculus was denser than in case R192, and was again localized to the same ventrolateral region contralateral to the injection. Dense anterograde labeling was seen over the entire pars compacts of the substantia nigra. Fewer labeled neurons were observed in the ventral tegmental area and the dorsal raphe

531

nucleus. The locus ceruleus contained a moderate amount of labeled cells on the ipsilateral side. No significant labeling was observed in the deep cerebellar nuclei. Slightly more retrograde labeling was seen in the medullary gigantocellular field than in R192. Case R210. The injection site in this case was centered rostral t o that of case R192 (Fig. 3). At rostral levels, the injection site overlaps with that of R205, and included part of retrorubral field, although t o a lesser extent than in the previous case. At caudal levels, the injection site included a small area of PPT. The injection site does not include the PPT pars compacta or the parabrachial nucleus. Overall labeling was very light throughout the brain. Moderately dense labeling was observed in the central tegmental field and periaqueductal gray. Moderate labeling was observed in the lateral habenula and in the ventral tegmental area. Analysis of labeling is presented in Table 3. The labeling observed in this case is significantly different from that of the other MEA cases. Fewer neurons were labeled in the lateral habenula, but labeling was similar to that seen in the other cases. In the forebrain, anterograde labeling was sparse in the striatum and globus pallidus, and no retrogradely labeled neurons were seen. In the hypothalamus, labeled neurons were observed in the medial parvicellular division of the paraventricular nucleus. There was significantly less labeling in the lateral hypothalamic area in this case. No significant labeling was observed in the anterior region. At posterior levels of LHA, labeled neurons were located adjacent to the cerebral peduncle. There were fewer labeled neurons in the dorsal raphe. In the parabrachial nucleus, additional labeling was observed in the external lateral and external medial subnuclei. Anterograde labeling in the substantia nigra pars compacta was relatively dense, and was concentrated over its central region, with medial and lateral regions less dense. In the locus ceruleus, retrograde labeling was light to moderate ipsilaterally.

Injections into other regions Case R194: parabrachial nucleus. The injection site in this case is centered in the rostral parabrachial nucleus, near the border between the PPT and PB (Fig. 4).The bulk of the injection encompasses the medial subnucleus of PB. At rostral levels, the injection site involves the caudal PPT and the ventrolateral aspect of the MEA. At caudal levels, the injection site surrounds the ascending superior cerebellar peduncle. Much of the labeling is found in structures such as the lateral hypothalamic area, periaqueductal gray, central tegmental field, dorsal raphe, and brainstem reticular formation. Analysis of labeling is presented in Table 4. Telencephalon. In the cortex, the rostral cingulate cortex was unlabeled; however, dense labeling was observed in the infralimbic cortex. The bed nucleus of the stria terminalis was densely labeled, mainly in the anterolateral, ventromedial, and caudal preoptic subnuclei (Moga et al., '89). This labeling blended into heavy retrograde labeling in the substantia innominata (SI), which was further contiguous with dense retrograde labeling in the central nucleus of the amygdala (CeA) where it was concentrated in its medial subdivision. This continuum of retrogradely labeling through the BST, SI, and CeA has been previously recognized following injections into the parabrachial area (Jackson and Crossman, '81a; De Olmos et al., '85; Moga et al., 'go), in part supporting anatomists who have long emphasized the commonality of these three regions on the basis of


532 TABLE 4. Analysis of Retrograde Labeling Following Injections in Adjacent Areas' Case no. (primary locus)

R194 (PB) Telencephalon Prefrontal cortex Septumidiagonal band Bed nucleus of the stria terminalis Central nucleus of the amygdala Substantia innorninata Diencepbalon Medial preoptic area Lateral preoptic area Paraventricular nucleus Lateral hypothalamic area, anterior Lateral hypothalamic area, tuberal Lateral hypothalamic area, posterior Posterior hypothalamic area Perifornical area Lateral habenula Zona incerta Mesencephalon Sunerior colliculus Substantia nigra, pars compacta Suhstantia nigra, pars reticulata Substantia nigra, pars lateralis Central tegmental field Ventral tegmental area Retrorubrd field Edinger-Westphal nucleus Central superior raphe Periaqueductal gray, dorsal Periaqueductal gray, lateral Periaqueductal gray, ventrolateral Pons Dorsal raphe nucleus Median raphe nucleus Cuneiform nucleus Laterodorsal tegmental nucleus Parabrachial nucleus, lateral Parabrachial nucleus, medial Locus ceruleus Barrington's nucleus Pontine tegmental field Gigantocellular tegmental field Cerebellar nucleus, lateral Cerbellar nucleus, interposed Medulla Nucleus prepositus hypoglossi Vestibular nuclei Nucleus of the solitary tract, rostral Nucleus of the solitary tract, caudal Spinal sensory trigeminal nucleus Medullary.ziaantocellular field _I Ventrolateral medulla

R189 (RRF)

+I-

++/+++I-

++++/+++I-

++I+ +I-

R198 (PTF)

++I-

-I-I-

++++ ++++I-

-I-I+I-

++++\+++I-

++++

+++

+I-

-I-/-

++I-

++I-

-I-

+++/+

+++I+ +++I+ +I-I+I+I-

+/++ ++/++I+I-

++++I+

++++I+

+I-I++I+ -I-

+++I+ +I-

+++I+ -I-

+I+I-

+I-

+ + +I-

++++I+

++++

+

*I;II+ *I+/-

++/-

++I+ +++I++ ++I+I-

+I+ +I+/-

+++ +I+

++I+

+i-

+++ +/-

++++/++

++++I+

+/-

++++I+ ++I+I-1+++I +

+I*I+++

+ ++

-I-

++++I++

-I+*I+

+ -

++I+

+++I+ ++++I+++

+++ 1 +

++++I++

++I+

+

+++ -

-

++I+

++/+

++++I++ +++I+ +I+I+++I++ ++I+ +++I+

+++*I+++ +++I+++

+I+ +I+ +;Il-I-1-

+I-

-I-I-

+I+ +I-

+I+ +I+

+++

-I-

+/-

-I+ +I-

++I+

-I+I++

+++I+++

++ji

'Semiquantitative analysis of retrograde labeling in brain regions following injections centered outside the pedunculopontine nucleus and midbrain extrapyramidal area, in the parabrachial nucleus (case R194), the retrorubral field (case R1891, and t h e pontine tegmental area (caseR198). The injection sites in these cases are depicted in Figure 4, and described in the text. Retrograde labeling is indicated as in Table 2.

cytoarchitecture, immunocytochemistry, and connections (De Olmos et al., '85; Holstege et al., '85; Moga et al., '89; Heimer et al., '90). Diencephalon. In the hypothalamus, moderate labeling was found in the medial preoptic area, with several labeled neurons located in the medial preoptic nucleus. The paraventricular nucleus was heavily labeled, with labeled neurons located within both the medial and lateral parvicellular regions. In the lateral hypothalamic area, labeling was dense in the anterior region, as well as in tuberal and posterior regions. Labeling in the posterior region was concentrated laterally and formed a "cap" around the

internal capsule. This labeling pattern was observed in earlier studies utilizing retrograde tracing in the parabrachial region (Jackson and Crossman, '81a; Grove, '88b; Moga et al., '901, substantia innominata (Grove, '88a), and central nucleus of the amygdala (Krettek and Price, '78; Saper et al., '79; Grove, '88b). In the thalamus, anterograde labeling was observed in midline and centromedian nuclei. Fewer labeled neurons were seen in the parafascicular nucleus than following injections into the PPT. A few labeled neurons were observed in the lateral habenula. Mesencephalon. Dense retrograde labeling was observed in the central tegmental field, the periaqueductal gray, and the dorsal raphe nucleus. Significantly less retrograde labeling was observed in the superior colliculus than following PPT injections. Light labeling was seen in the SN pars compacta and reticulata. A few labeled neurons were seen in the dorsal nucleus of the lateral lemniscus, paralemniscal nucleus, and MEA on the contralateral side. Labeling in the retrorubral field was moderately dense. Pons. Labeling observed in Barrington's nucleus was relatively dense. In the cerebellar nuclei, the lateral nucleus was moderately labeled, the interposed nucleus contained several labeled neurons, and the medial nucleus was unlabeled. A few retrogradely labeled neurons were seen bilaterally in prepositus hypoglossi. Labeling in the pontine and medullary reticular formation was comparable t o that seen in PPT injections. Case R189: retrorubral field. The injection site in this case was centered in the RRF at its midcaudal level (Fig. 4). At rostral levels, the injection site involved the central tegmental field lateral to the red nucleus. At middle levels, the injection site involved the rostral PPT, the ventral central tegmental field, and the medial portion of the paralemniscal nucleus. At caudal levels, the injection site involved the ventral PPT and part of the MEA. The injection site avoids the dorsal nucleus of the lateral lemniscus, PPT pars compacta, cuneiform nucleus, and parabrachial nucleus. Analysis of labeling is presented in Table 4. Telencephalon. In prefrontal cortex, the rostral cingulate and agranular insular regions were unlabeled. The infralimbic cortex was densely labeled. The nucleus accumbens was heavily labeled, as was the ventral tail of the striatum (fundus striati). Dense labeling was observed in the bed nucleus, the central nucleus of the amygdala, and the substantia innominata. The pattern of retrograde labeling in these regions is remarkably similar to that observed in R194. Additional substantia innominata labeling was observed ventral t o the globus pallidus. Diencephalon. Dense labeling was observed in the paraventricular nucleus and lateral hypothalamic area. The pattern of labeling in LHA was quite similar to that seen in R194. Again, a dense cluster of labeled neurons was observed surrounding the internal capsule and cerebral peduncle. In the thalamus, anterograde labeling was seen in midline and intralaminar nuclei, and very slight anterograde labeling was observed in the lateral geniculate. In the posterior thalamus, several labeled neurons were observed in the parafascicular nucleus. A few labeled neurons were seen in the entopeduncular nucleus and in the lateral habenula. Mesencephalon. Dense anterograde labeling was observed in the central tegmental field. Labeled neurons were found diffusely throughout the region. Moderately dense labeling of the periaqueductal gray was observed. The


533

superior colliculus was moderately labeled, and the pattern was similar to that seen in PPT injections. In the periaqueductal gray, the dorsal subdivision was densely labeled. Retrograde labeling in the lateral quadrant was moderately dense, and more labeling was observed contralaterally. The contralateral RRF and PPT were moderately labeled. A few labeled neurons were seen in the DLL bilaterally. Very few labeled neurons were observed in the contralateral MEA. Pons. In the parabrachial nucleus, retrograde labeling was dense in the lateral and medial subnuclei. Moderate labeling was observed in the superior lateral, central lateral, and dorsal lateral subnuclei. Labeling was not observed in the external lateral or Kolliker-Fuse subnuclei. A few labeled neurons were seen in the locus ceruleus, and labeled neurons were also observed in the pontine central gray, ventral to the rostral LC. An additional cluster of labeled neurons was observed in the pontine central gray, rostral and dorsolateral to the genu of the facial nerve. A few labeled neurons were observed in Barrington's nucleus. Labeling seen in the pontine reticular formation and medulla was similar to that seen in case R194, except fewer labeled neurons were observed in the caudal nucleus of the solitary tract. Case Rl98: pontine tegmental field. The injection site in this case is centered in the pontine tegmental field, ventral t o the PPT (Fig. 4). The injection site extends as far rostral as the retrorubral field. Dorsally, the injection site slightly involves the PPT, but avoids the nuclei of the lateral lemniscus, laterally, and the median raphe, medially. Caudally, the injection site is located ventral to the medial parabrachial nucleus. Analysis of labeling is presented in Table 4. Telencephalon. No significant labeling was seen in the cerebral cortex. A few labeled neurons were seen in the bed nucleus, which were located in the ventromedial and caudal preoptic subnuclei. The amygdala was unlabeled. The substantia innominata contained a few labeled neurons in the ventral pallidal region. Diencephalon. The medial preoptic area was unlabeled, and few labeled neurons were seen in the lateral preoptic area. Light labeling of the parvicellular paraventricular nucleus was observed. Lateral hypothalamic labeling was relatively light, and was observed at tuberal and posterior levels. A few labeled neurons were seen in dorsomedial nucleus. In the thalamus, light anterograde labeling was seen in midline and intralaminar nuclei. A few labeled neurons were seen in the parafascicular nucleus. Moderately dense labeling was seen in the zona incerta, where labeled neurons were concentrated medially at rostral levels. Mesencephalon. Very dense labeling was observed in the central tegmental field. Dorsal and lateral periaqueductal gray was densely labeled. In the ventrolateral quadrant, a few labeled neurons were seen at the ventral edge. A moderate number of labeled neurons was observed in the deep layers of the superior colliculus. Very little labeling was seen in the substantia nigra; a few labeled neurons were seen in the pars reticulata. A few labeled neurons were seen in the dorsal raphe and in the cuneiform nucleus. Pons. In the rostral parabrachial nucleus, labeling was partially obscured by labeled fibers extending from the injection site. No labeling was observed in the caudal parabrachial nucleus, locus ceruleus, or Barrington's nucleus. A cluster of labeled neurons was observed in the pontine central gray, rostral and dorsolateral to the genu of

the facial nerve. No significant labeling was seen in the deep cerebellar nuclei. The pontine reticular formation was densely labeled bilaterally. Medulla. All medullary reticular fields were labeled. The gigantocellular field was densely labeled, and several labeled giant cells were observed. Labeling in the dorsal reticular formation was light to moderate. The contralatera1 spinal trigeminal nucleus contained several labeled neurons that were located ventrally. A few labeled neurons were observed in the prepositus hypoglossi. No labeling was observed in the solitary nucleus.

DISCUSSION Technical considerations The possible uptake of retrograde tracers by fibers of passage precludes the definitive identification of afferents, especially in the case of the reticular formation, where fiber pathways are abundant. Therefore, the afferents identified in this study are a first approximation only, and remain putative afferents until they can be confirmed in further studies using anterograde tracing techniques. However, the results of several studies suggest that the tracer utilized in this study, peroxidase-conjugatedWGA, can be taken up by damaged fibers, but is not significantly transported by intact fibers of passage. For example, Brodal et al. ('83) found no appreciable transport by intact fibers following pressure injections of 0.4-0.6 ~1 of 2% WGA-HRP into the pons. Similarly, radiolabeled WGA injected into subcortical white matter was not significantly transported (Steindler, '82; Grob et al., '82). In addition, the results of our studies demonstrate unique labeling patterns following closely placed injections. For example, the LHb is robustly labeled from injections into the MEA, but is relatively unlabeled following injections into the adjacent PPT (Fig. 10). This difference is more likely due to the specific localization of the injection than to tracing from fibers of passage. These findings argue against the fibers-of-passageproblem being a significant source of false positives or negatives. Nonetheless, in further studies we will utilize anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L) combined with ChAT immunohistochemistry to confirm the afferents to the PPT and MEA that have been identified in the present study. Preliminary results of anterograde tracing from the lateral hypothalamus and dorsal raphe nucleus have been reported (Steininger and Wainer, '90, '91). Anterogradely labeled projections to the PPT and/or MEA will be examined at the ultrastructural level to confirm synaptic innervation. The results of these studies will be presented in subsequent reports in this series.

General observations The results of the present study, as summarized in Figures 11 and 12, have indicated that the PPT and MEA share several putative afferent sources such as the lateral hypothalamic area, periaqueductal gray, dorsal raphe, and pontine reticular fields. There nevertheless seems to be subnuclear organization within several of these afferent sources, which is reflected in their differential projections to either the PPT or MEA. The present study has identified many more brain regions with patterns of efferent connectivity, which appears to support a distinction between the PPT and MEA more clearly. The results of the present study, for example, support our previous investigations that ascribe the afferent and efferent connections of the

534

Fig. 10. Photomicrographs taken under polarization optics. A , B Retrogradely labeled neurons observed in the lateral habenula following retrograde tracer injections into the midbrain extrapyramidal area (case R192) (A) and pedunculopontine tegmental nucleus (case R220) (B). The ventrolateral edge of the lateral LHb is indicated by arrows. C,D: Retrogradely labeled neurons and anterogradely labeled fibers observed in the entopeduncular nucleus (arrow in C) following tracer injections into the midbrain extrapyramidal area (case R192) (C) and


pedunculopontine tegmental nucleus (case R220) (D).The corresponding region is indicated by the arrow in D. Labeling in the lateral hypothalamus is indicated by the arrowhead in D. E,F: Anterogradely labeled fibers observed in the substantia nigra pars compacta (arrow in E) followingretrograde tracer injections into the midbrain extrapyramiand pedunculopontine tegmental nucleus (case dal area (case R192) (E) R220) (F).The corresponding area is indicated by the arrow in F. Scale bar = 800 pm.


535

PPT Fig. 11. Summary of afferents to the pedunculopontine tegmental nucleus, as discussed in the text, are illustrated in this schematic horizontal section.

Fig. 12. Summary of afferents to the midbrain extrapyramidal area, as discussed in the text, are illustrated in this schematic horizontal section.

mesopontine tegmentum with the basal ganglia to the MEA, and not the PPT. We have extended these findings by identifying patterns of connectivity of the lateral habenula and superior colliculi with the mesopontine tegmentum that are unique to the MEA and that probably reflect an extension of the aforementioned traditional extrapyramidal circuitry. Several putative afferents to the PPT not shared with the MEA include several medullary structures, in particular the caudal NTS and the gigantocellular and ventrolateral medullary fields. We have also identified the patterns of afferent connectivity putatively ascribed to either the MEA or PPT that distinguish them from surrounding structures in the mesopontine tegmentum. In this process we have determined that the retrorubral field and parabrachial nuclei, between which the PPT and MEA are sandwiched, display a remarkable homogeneity in afferents. It is difficult to compare the results of our present studies to previous investigations, as most were performed without awareness of the nuclear organization of the mesopontine tegmentum, which has been gradually revealed over the last 7 years through correlative analyses of cytoarchitectonic, chemoarchitectonic, and connectional data (Fulwiler and Saper, '84; Rye et al., '87). In the majority of previous studies, for example, the PPT is not recognized as it is here, as a cholinergic nucleus with distinct connections from the

adjacent MEA. Rather, the PPT has been most frequently considered a larger, more heterogeneous region of the mesopontine tegmentum that occupies a "peribrachial" position; therefore confusion is frequently engendered with the parabrachial nucleus. These issues are further confounded by significant interspecies differences in the chemoarchitecture of the mesopontine tegmentum (Rye et al., '87; Jones and Beaudet, '87; Henderson, '87). Therefore, when comparing the results of the present study with others discussed below, we can find support for all the projections identified here when we consider the literature as a whole. Retrograde studies, for example, have identified projections to the mesopontine tegmentum from the preoptic area (Swanson et al., '84, '871, hypothalamus (Pare et al., '89; Jackson and Crossman, '81a; Moon-Edley and Graybiel, '83; Moriizumi et al., '88; Fibiger and Semba, '881, substantia innominata (Moon-Edley and Graybiel, '83; Moriizumi et al., '881, amygdala (Jackson and Crossman, '81a; Moon-Edley and Graybiel, '83; Fibiger and Semba, '88; Moriizumi et al., '881, zona incerta (Watanabe and Kawana, '82; Moriizumi et al., '88; Fibiger and Semba, '881, ventral tegmental area (Jackson and Crossman, 'Sla), midbrain reticular formation (Moriizumi et al., '88; Pare et al., '901, periaqueductal gray (Jackson and Crossman, '81a; Moriizumi et al., '88; Fibiger and Semba, '88; Pare et al., '901, dorsal raphe (Moriizumi et al., '88; Fibiger and Semba,


536

'881, pontine and medullary reticular formation (MoonEdley and Graybiel, '83; Fibiger and Semba, '88; Pare et al., '901, superior olive (Moon-Edley and Graybiel, '831, spinal trigeminal nucleus (Pare et al., '901, and nucleus prepositus hypoglossi (Higo et al., '90). Similarly, investigations of these pathways with anterograde technologies are supportive but rarely definitive as it is difficult to extrapolate other results to the nuclear scheme employed in our analysis. Anterograde projections to the mesopontine tegmentum have been identified with 3H amino acids from the lateral hypothalamic area (Hosoya and Matsushita, '81; Berk and Finkelstein, '821, zona incerta (Ricardo, '81; Watanabe and Kawana, '821, and pontine and medullary reticular formation (Vertes and Martin, '88; Graybiel, '77; Loewy et al., '81; Vertes et al., '86) and with PHA-L from paraventricular nucleus (Luiten et al., '851, nucleus prepositus hypoglossi (Vertes and Martin, '881, and medial prefrontal cortex (Sesack et al., '89).

Afferents common to the PPT and MEA Moderately dense projections from the superior colliculus to the PPT and MEA were observed. However, the projection to the MEA originates from a different, distinct region of the SC that gives rise to a descending pathway separate from the projection to the PPT. Following injections into the MEA, retrograde labeling was observed contralaterally, in a lateral region of the intermediate deep layer, whereas following injections into the PPT, labeling was observed primarily ipsilaterally, from neurons scattered throughout the deep layers. Anterograde and retrograde tracing studies in the cat (Kawamura et al., '74; Graham, '77; Edwards, 'SO), monkey (Harting, '77), rabbit (Holstege and Collewijn, '821, and rat (Redgrave et al., '87; Dean et al., '88) have identified two main descending fiber systems and their origins in the tectum: 1) an uncrossed projection originating in most of the deep layers and terminating densely in the lateral reticular formation, as well as in the pontine nuclei and reticulotegmental nucleus; and 2) a crossed projection arising from neurons in the intermediate white layer that crosses the midline in the dorsal tegmental decussation, turns caudally to run in the predorsal bundle, and terminates densely in the medial reticular formation, and less densely in oculomotor and perioculomotor structures, precerebellar nuclei, and spinal cord. Interestingly, it is this intermediate deep layer that is in receipt of afferents from the substantia nigra pars reticulata (May and Hall, '84). These two pathways represent functionally distinct output channels as indicated by the results of stimulation and lesion studies (for references see Dean et al., '88). The crossed pathway is likely to mediate orienting behaviors (i.e., contralaterally directed head and eye movements to visual and/or other sensory stimuli), whereas the uncrossed pathway is likely to mediate defensive reactions such as freezing, retreat, or escape. The periaqueductal gray was found to provide dense input to both the PPT and the MEA, as well as to the parabrachial nucleus and retrorubral field. This projection was found to originate mainly in the lateral and ventrolateral quadrants. The results of anterograde tracing studies in the cat (Hamilton, '73; Ruda, '7.9, monkey (Mantyh, '831, and rabbit (Meller and Dennis, '91) indicate that the densest tegmental projections arise from the lateral and ventrolateral PAG. The PAG has long been implicated in

pain control systems and as a site for stimulus-produced analgesia (for review see Basbaum and Fields, '84). The most effective stimulation sites are found in the ventrolateral quadrant (Gebhart and Toleikis, '781, where enkephalinimmunoreactive neurons and terminals are concentrated (Moss et al., '83). However, the PAG is also implicated in complex defensive behaviors ("flight or fight"); therefore, projections from the PAG may mediate the behavioral and physiological changes associated with this state, including locomotion and/or defensive postures, cardiovascular responses, analgesia, and arousal (Abrahams et al., '60; Hilton and Redfern, '86; Bandler and Carrive, '88; Depaulis et al., '88). A substantial input to both the PPT and MEA originates from a compact subregion of the dorsal raphe, in the dorsal part of the nucleus. Anterograde autoradiographic studies in the cat (Bobillier et al., '76; Pierce et al., '76) have depicted labeled fibers in the PPTiMEA region following tracer injections in DR, although a detailed examination of these tegmental projections was not performed. In preliminary studies, we have utilized anterograde tracing with PHA-L combined with ChAT immunocytochemistry to demonstrate that the DR provides dense innervation of the PPT and MEA (Steininger and Wainer, '91). A dense projection from the central tegmental field to the PPT was observed bilaterally, with an ipsilateral dominance. The CTF projection to the MEA was much less dense, and was only observed caudal to the red nucleus. Projections from the pontine reticular formation to the PPT originate mainly from medial regions of the rostral PTF, with lesser projections from the caudal GTF. In lateral regions, retrogradely labeled neurons were more dispersed. A lesser projection goes to the MEA, which is is observed mainly at rostral levels. Numerous tracing studies have determined that the medial pontine reticular formation (mPRF) receives cholinergic innervation from the PPT and LDT (Mitani et al., '88; Shiromani et al., '88; Quattrochi et al., '89; Rye et al., '88; Jones, '90). Following injections into the PPT, retrogradely labeled neurons were observed throughout the medullary reticular formation, but were especially prominent in the dorsomedial region, rostrally, and in the ventrolateral region, caudally. The labeled region in the ventrolateral medulla, lateral to the pyramids, contains the A1 and C1 cell groups (Armstronget al., '82; Ruggiero et al., '85). Following injections into the MEA, labeled neurons were observed only rostrally, whereas, they were located contralaterally in the dorsomedial region. Lateral hypothalamic afferents are common to both PPT and MEA, although the pattern of labeling in MEA injections is different from that seen in PPT injections. Injections into the PPT labeled neurons in LHA that are somewhat restricted to the lateral portion of LHA, whereas more labeled neurons are located diffusely in the dorsal and medial regions of LHA, and also in perifornical regions following injections into the MEA. In addition, labeling of the anterior level was greater following MEA injections, and labeling of the posterior level was greater following PPT injections. The LHA is known to participate in a variety of complex behaviors including emotional response (Smith and DeVito, '841, arousal (Shoham and Teitelbaum, '82; Lin et al., '891, and goal-directed locomotion (Markowska et al., '85; Blake and Gladfelter, '86; Sinnamon and Stopford, '87; Lammers et al., '88; Marciello and Sinnamon, '90).


Afferents to the PPT

537 that rostral injections involved more of the surrounding regions (i.e.,central tegmental field, retrorubral field).

In the present study, the retrograde transport of WGAHRP revealed putative afferents to the PPT from wideAfferents to the MEA spread brain regions. The results of this study indicate that The results of retrograde tracing from the MEA indicate the major inputs to the PPT originate in the periaqueductal gray, the central tegmental field, dorsal raphe, lateral that this region receives a major input from the lateral hypothalamic area, superior colliculus, and brainstem retic- habenula, as well as dense projections from the lateral ular formation. Less prominent inputs seem to originate in hypothalamus, periaqueductal gray, and dorsal raphe nuthe paraventricular and preoptic hypothalamic nuclei, zona cleus. Moderate afferents originate in extrapyramidal moincerta, nucleus of the solitary tract, central superior raphe tor system structures including the substantia nigra and nucleus, substantia innominata, posterior hypothalamic entopeduncular nucleus, the ventral tegmental area, and area, and thalamic parafascicular nucleus. Retrograde label- the substantia innominata. Extrapyramidal motor system. Tracing studies in the ing in structures such as the bed nucleus of the stria terminalis, central nucleus of the amygdala, substantia rat (Jackson and Crossman, '81a,b; Saper and Loewy, '82; innominata, and paraventricular nucleus was most likely Takada et al., '88) and cat (Moon-Edley and Graybiel, '83; due to the involvement of the parabrachial nucleus or Larsen and McBride, '79; Moriizumi et al., '88) have used retrorubral field in the injection sites. Dense labeling in the terminology of Olszewski and Baxter ('54) on a topologthese structures following injections centered in the retroru- ical, rather than cytological, basis to define the "nucleus bra1 field and parabrachial nucleus became progressively tegmenti pedunculopontis" as the region that receives less prominent as injections centered themselves in the basal ganglia and related afferents. In a previous study we PPT. However, in the present study it could not be deter- performed a cytoarchictectonic study of the rat PPT, and mined whether these regions also innervate the PPT. This the relationship of nigral and pallidal terminations to the cytoarchictectonically defined PPT was examined (Rye et question will be addressed in future studies employing al., '87). Th'e results of this study determined that nigroanterograde tracing combined with ChAT immunohis- tegmental and pallidotegmental efferents terminate in a tochemistry. Similarly, following injections into the RRF or small-celled region of the central tegmental field adjacent to PB, a dense "cap" of retrogradely labeled cells was observed the cholinergic PPT, which was then termed the MEA. In in the posterior lateral hypothalamic area, along the medial the monkey, cat, and rat, afferents to this region have been edge of the subthalamic nucleus; it was observed only in reported from the subthalamic nucleus (Jackson and Crossinjections involving the RRF or PB. Utilizing anterograde man, '81b; Kita and Kitai, '87; Moriizumi et al., '88; Takada tracing with PHA-L, we have determined that this region et al., '88; Granata and Kitai, '89; Smith et al., 'go), the projects to both the RRF and PB, but contributes only entopeduncular nucleus (primate internal pallidal segslightly to the innervation of the PPT (Steininger and ment) (Larsen and Sutin, '78; Filion and Harnois, '78; Wainer, '90). Larsen and McBride, '79; Van der Kooy and Carter, '81; The projection from the nucleus of the solitary tract to Harnois and Filion, '82; Moriizumi et al., '88), and the the parabrachial complex has been well characterized (Nor- substantia nigra pars reticulata (Noda and Oka, '84, '86; gren, '78; Herbert et al., '90; Herbert and Saper, 'go), and Beckstead et al., '79; Beckstead and Frankfurter, '82; while it is possible that labeling in the caudal NTS following Moriizumi et al., '88; Spann and Grofova, '91; Nakamura et PPT injections may reflect the involvement of the parabra- al., '89). chial nuclei in the injection site, it seemed unlikely for In the present study we have demonstrated afferents to several reasons. First, labeling of the caudal NTS appears in the MEA from the entopeduncular nucleus and substantia all cases with injections centered in the PPT, including nigra pars reticulata. The pars reticulata was also labeled injections that avoid the parabrachial nucleus. Second, following injections into the PPT that involved the MEA NTS labeling is similar in all PPT injections, with the (case R220), but was sparse in other PPT injections. We did exception of the rostral part of the NTS, which is lightly not observe a projection from the subthalamic nucleus in labeled in case R220, but is virtually unlabeled in other PPT any of the MEA injections. The existence of a subthalamicinjections. Confirmatory evidence comes from observations tegmental pathway is a matter of controversy in the that injections of the anterograde tracer PHA-L into the literature. Anterograde tracing from the STh in several medial NTS produces anterograde labeling rostrul to PB, in species has yielded conflicting results. A weak projection the region occupied by the caudal PPT (Herbert et al., '90; was observed in the cat (McBride and Larsen, '80; Moonpersonal observations). Edley and Graybiel, '83; Moriizumi et al., '881, monkey We have utilized injections into different rostrocaudal (Nauta and Cole, '78; Smith et al., '901, and rat (Kita and regions of PPT to assess the afferents to this nucleus. Our Kitai, '87; Jackson and Crossman, '81a); however, no results indicate that afferents may be topographically orga- projection was observed in other studies performed in nized with respect to the rostrocaudal axis of the nucleus. monkey (Carpenter et al., '81; Carpenter and Jayaraman, The rostral PPT appears to receive more input from the '901, cat (Nauta and Cole, '78), or rat (Ricardo, '80).Several substantia innominata, paraventricular hypothalamic nu- investigators have suggested that anterograde labeling cleus, and anterior lateral hypothalamic area, and less observed in the tegmentum may have resulted from the input from the tuberal and posterior lateral hypothalamic diffusion of tracer into the adjacent zona incerta or lateral area and dorsal raphe nucleus. Further studies are needed hypothalamic area (Smith et al., '90; Kita and Kitai, '87; to determine whether these projections are topographically Carpenter et al., '81). Retrograde labeling in the STh was organized, or whether the observed differences in retro- observed followinglarge injections of tracer into the tegmengrade labeling are due to the fact that fewer cholinergic tum ofthe rat (Jackson and Crossman, '81a,b; Hammond et neurons are located in rostral regions of the nucleus, and al., '83; Takada et al., '88). The population of labeled STh

538 neurons was estimated as 1%of the total STh population that included a lateral region outside the nucleus proper (Hammond et al., '83; Takada et al., '88). Based on the results of the present study, it is unlikely that the STh contributes significantly to the innervation of the MEA. We have also observed dense anterograde labeling in the substantia nigra pars compacta, and moderate anterograde labeling in the entopeduncular nucleus, caudate putamen, globus pallidus, and subthalamic nucleus following tracer injections into the MEA. This confirms the results of a previous tracing study in our laboratory (Lee et al., '881, and other tracing studies in the rat (Saper and Loewy, '82; Jackson and Crossman, '83; Hammond et al., '83; Scarnati et al., '86; Canteras et al., '90) and cat (Rinvik et al., '79; Nomura et al., '80; Gonya-Mageeand Anderson, '83; MoonEdley and Graybiel, '83). The MEA projection to the pars compacta is particularly striking, when compared to more traditional extrapyramidal inputs to the pars compacta, which occur predominantly on distal dendritic arbors in the pars reticulata (Fallon and Loughlin, '85). One might therefore expect that this focused projection on somata of pars compacta neurons would place the MEA in a position to influence profoundly dopaminergic output neurons of the nigra. Lateral habenula. The major input to the MEA originated in the lateral subdivision of the lateral habenula. The division of the LHb into medial and lateral regions is based on the differential afferent and efferent connections of these regions (Herkenham and Nauta, '79). The medial LHb receives most of its afferents from limbic regions and projects mainly to the midbrain raphe nuclei (Herkenham and Nauta, '79). The lateral LHb is afferented mainly by the entopeduncular nucleus (Herkenham and Nauta, '77; Nauta, '74; Larsen and McBride, '79; Parent, '79) and was found to project predominantly to a large region of the medial reticular formation that is traversed by decussating fibers of the superior cerebellar peduncle (Herkenham and Nauta, '79). Herkenham and Nauta ('79) also note that this projection exhibits a striking increase in grain density in its terminal field, suggesting a more profuse and concentrated preterminal fiber arborization, which would account for the extreme density of retrograde labeling in the lateral LHb observed in the present study.

T.L. STEININGER ET AL. the medullary reticular formation, gigantocellular tegmental field, central tegmental field, and zona incerta. Moderate labeling was observed in the periaqueductal gray, the cingulate cortex, and the contralateral pontine tegmental field. The exuberance of reticular interconnections has been noted in previous tracing studies (Gallager and Pert, '78; Jones andYang, '85; Shammah-Lagnado et al., '87).

Functional considerations

Locomotor activity. The region of the PPTIMEA has been identified as the mesencephalic locomotor region (MLR). Stimulation of the MLR elicits treadmill locomotion in postmammillary-precollicular transected animals (Shik et al., '66; Grillner and Shik, '73; Skinner and Garcia Rill, '84). The stimulation parameters used in these studies preclude the precise anatomical localization of the MLR, because of the spread of current away from the stimulation site. The classical description of the MLR is the region of cunieform nucleus, dorsal PPT and lateral PAG. A recent study localized the MLR by reconstruction of the lowest threshold sites required for locomotion (Coles et al., '89). The location of the MLR as determined by this study more closely corresponds to the cuneiform nucleus dorsal to the PPT and MEA. However, induced locomotor activity may not be specifically related to pathways normally active in generating locomotion, as the transections employed in these studies introduce a high level of background excitability in spinal locomotor systems (Mori et al., '86). This nonspecific nature of induced locomotion is indicated by the induction of locomotion in the mesencephalic preparation by stimulation of regions such as the cochlear nuclei, cuneate nucleus, and spinocerebellar tract, which are outside of known locomotor regions (Beresovskii and Bayev, '88). The functional role of the MEA is as yet unclear. Given the nature of the putative afferents from: 1)extrapyramidal motor structures such as the lateral LHb; 2) regions implicated in complex behaviors such as LHA and PAG; 3) regions implicated in arousal processes such as DR and LHA; and also given the close association with the PPT, it appears likely that the MEA may participate in the modulation of motor systems in relation to the behavioral state of the animal through efferents to extrapyramidal motor system structures such as the dense projection to the pars Afferents to surrounding regions compacta of the substantia nigra. Arousal and sleeplwakefulness. The PPT is believed to Parabrachial nucleus and retrorubral field. In the present study, we have described afferents to the parabra- participate in the modulation of behavioral states. Electrochial nucleus from the prefrontal cortex, preoptic area, bed physiological studies have determined that the neurons of nucleus of the stria terminalis, central nucleus of the the PPT exhibit state-dependentactivation, such that firing amygdala, substantia innominata, posterior lateral hypotha- rates are highest during waking and REM sleep, and lower lamic area, dorsal raphe, periaqueductal gray, brainstem during slow-wave sleep (El Mansari et al., '89). The efferent reticular formation, and nucleus of the solitary tract. Our connections of the cholinergic brainstem nuclei are consisfindings in the present study are consistent with previous tent with this hypothesis. Ascending projections to the reports of PB afferent connections (Moga et al., '90; Her- thalamus (Hallanger et al., '87; Hallanger and Wainer, '88) bert et al., '90). We have also demonstrated that many PB facilitate thalamocortical transmission (Dingledine and afferents are shared by the retrorubral field (i.e., the Kelly, '77; Marshall and Murray, '80; Sillito et al., '83; BST-SI-CeA"continuum" and the posterior lateral hypotha- McCormick and Prince, '86; Francesconi et al., '88; McCorlamic "cap" adjacent to the internal capsule and cerebral mick and Pape, '88; Marks et al., '891, which leads to the peduncle) and that these two regions are reciprocally cortical desynchronization that is characteristic of waking connected, indicating that a close relationship exists be- and the REM sleep state. Projections to the hypothalamus tween the PB and the RRF. and basal forebrain may function indirectly in cortical Pontine tegmental field. Retrograde tracing from the activation via diffuse cortical projections (Rye et al., '84b; pontine tegmental field ventral to the PPT revealed that Saper, '84, '85, '88). Descending projections of the PPT to inputs to this region originate mainly in other "reticular" the pontine and medullary reticular formation are believed fields. Dense retrograde labeling was observed bilaterally in to underlie the functions of the PPT in both tonic and

AFFERENTS TO THE MESOPONTINE TEGMENTUM phasic phenomena of REM sleep, including ponto-geniculooccipital (PGO) waves, rapid eye movements, and muscle atonia (Rye et al., '88; Mitani et al., '88; Jones, '90). PGO waves are biphasic field potentials that precede the appearance of other signs of REM sleep (i.e., cortical desynchronization, muscle atonia, and rapid eye movements) by 30-90 seconds (for review see Hobson and Steriade, '86; Steriade and McCarley, '90). Investigators initially detected PGO waves in the pontine reticular formation, lateral geniculate nucleus, and occipital cortex, although it is now known that PGO waves are not restricted to these regions (Steriade and McCarley, '90). Several experimental results have implicated the PPT as the final common path for the transfer of brainstem-generated ponto-geniculo-occipitalwaves to the thalamus: 1)lesions (Webster and Jones, '88; Sakai, '80)or reversible cooling (Laurent and Alayaguerrero, '75) of the PPT region suppressed or significantly diminished thalamic PGO waves; 2) microinjection of nicotinic agonists into the LGN decreased PGO waves (Hu et al., '88); and 3) a proportion of thalamic projecting PPT neurons discharge a burst of spikes that precede the detection of thalamic PGO waves by 10-25 ms (so called "PGO burst neurons") (McCarley et al., '78; Sakai and Jouvet, '80; Nelson et al., '83; Sakai, '85). The afferent projections to the PPT, as demonstrated in the present study, are consistent with the role of the PPT in behavioral state control. Reticular formation afferents may play a key role in the activation of PPT neurons. Electrophysiological studies have determined that the neurons in the central tegmental field fire tonically at higher rates during waking and REM sleep (Steriade, '81; Steriade et al., '82) and increase their firing rate several hundreds of milliseconds before the detection of thalamic PGO waves (Pare et al., '90). The medial pontine reticular formation has been strongly implicated in the generation of REM sleep: 1)electrophysiological recording studies have determined that neurons in mPRF exhibit state-dependent firing, with an increase in firing rate during the transition from slow wave to REM sleep (McCarley and Hobson, '75; Vertes, '79); and 2) microinjections of cholinomimetic drugs into mPRF elicit the physiologic components of REM sleep including PGO waves, rapid eye movements, muscular atonia (Quattrochi et al., '89; Baghdoyan et al., '84), and cardiorespiratory changes (Lydic et al., '91) that occur naturally during REM sleep; and 3) neurons of the medial pontine reticular formation increase their firing rate 50150 ms prior to thalamic PGO detection (Steriade and McCarley, '90; McCarley and Ito, '83). The results of these studies suggest that the activation of reticular formation neurons occurs prior to the activation of PPT neurons, and that projections from mPRF and CTF provide excitatory input to PPT neurons and may be required for the transfer of PGO waves to the thalamus. The dorsal raphe nucleus may have a profound effect on PPT neurons during sleepiwake cycles. The DR provides widespread serotonergic innervation to the forebrain, including the cerebral cortex and hippocampus (Bobillier et al., '76; Pierce et al., '76; Moore et al., '781, and exhibit state-dependent activation. Electrophysiological studies demonstrate that DR neurons decrease their firing rate during slow-wave sleep, cease firing 2-10 seconds prior to the onset of PGO waves, and are silent throughout REM sleep (McGinty and Harper, '76; Trulson et al., '81). Experimental results suggest that serotonin is inhibitory to PPT neurons in vitro (Leonard and Llinas, '90); therefore,

539

one might expect that the cessation of DR activity prior to REM sleep would lead to a disinhibition of PPT neurons and, consequently, the onset of thalamic PGO activity. This hypothesis is supported by pharmacologic evidence that depletion of serotonin in situ by systemic parachlorophenylalanine results in the paradoxical generation of thalamic PGO waves during waking (Dement et al., '72). A role for the posterior LHA in sleepiwakefulness has been reported in several studies. An electrophysiological study has demonstrated that neurons of the posterior LHA that project to the PPT have higher firing rates in waking than in slow wave sleep (Pare et al., '89). Chemical lesions of the posterior LHA result in an acute increase in REM sleep with no change in the amount of waking (Jouvet, '881, and microinjections of muscimol elicit a short-latencyinduction of slow-wave sleep followed by a significant increase of REM sleep (Lin et al., '89), suggesting that neurons of the LHA may tonically inhibit REM sleep.

Final comments It is interesting to note that Parkinson's disease (PD) and progressive supranuclear palsy (PSP),which both are characterized pathologically by a loss of PPT neurons (Hirsch et al., '87; Zweig et al., '87), share varying degrees of insomnia and motor dysfunction as clinical manifestations (Aldrich et al., '89). The nearly total loss of large, presumably cholinergic neurons of the PPT seems to be a more constant feature of PSP (Zweig et al., '87) than PD, in which cell loss of medially adjacent neurons (?MEA) is the predominant feature in some cases (Hirsch et al., '87). As our appreciation of a presumptive role for the PPT in REM-sleep generation becomes more well founded, this finding may account for why a more severe insomnia characterizes PSP. These observations, however, do not prove causality, and while we recognize that disease processes involving the mesopontine tegmentum certainly involve heterogeneous populations of neurons, we see a potential for careful clinicopathologic investigations in contributing to our further understanding of the functional importance of mesopontine cell groups in normal behavior.

ACKNOWLEDGMENTS The authors extend their thanks to Steven Price for his expertise in photographing and preparing figures for this manuscript, and Dr. C.B. Saper for helpful discussions. This work is supported by PHS NS17761 (B.H.W.) and PHS MH09919 (T.L.S.)

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Cholinergic and non-cholinergic neurons in the rat pedunculopontine tegmental nucleus.

Retrograde tracing of projections between the nucleus submedius, the ventrolateral orbital cortex, and the midbrain in the rat.

Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in the rat.

Descending brainstem projections of the pedunculopontine tegmental nucleus in the rat.

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

Topographical projections from the thalamus, subthalamic nucleus and pedunculopontine tegmental nucleus to the striatum in the Japanese monkey, Macaca fuscata.

Evidence for a cholinergic projection from the pedunculopontine tegmental nucleus to the rostral ventrolateral medulla in the rat.

Pedunculopontine tegmental nucleus neurons provide reward, sensorimotor, and alerting signals to midbrain dopamine neurons.

Separate neuronal populations of the rat globus pallidus projecting to the subthalamic nucleus, auditory cortex and pedunculopontine tegmental area.

Projections of the medial cerebellar nucleus to oculomotor-related midbrain areas in the rat: an anterograde and retrograde HRP study.

Critical role of cholinergic transmission from the laterodorsal tegmental nucleus to the ventral tegmental area in cocaine-induced place preference.

Ultrastructure of the mammillotegmental projections to the ventral tegmental nucleus of Gudden in the rat.

Collateral projections from the midbrain periaqueductal gray to the nucleus raphe magnus and nucleus accumbens in the rat. A fluorescent retrograde double-labelling study.

Autoradiographic studies of the projections of the midbrain reticular formation: ascending projections of nucleus cuneiformis.

The development of ventral tegmental area (VTA) projections to the visual cortex of the rat.

REM sleep diversity following the pedunculopontine tegmental nucleus lesion in rat.

Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat.

Afferent connections to the amygdaloid complex of the rat and cat. I. Projections from the thalamus.

Sources of input to the rostromedial tegmental nucleus, ventral tegmental area, and lateral habenula compared: A study in rat.

Cerebellar nuclei and the nucleocortical projections in the rat: retrograde tracing coupled to GABA and glutamate immunohistochemistry.

Ultrastructure of cholinergic neurons in the laterodorsal tegmental nucleus of the rat: interaction with catecholamine fibers.

Firing of 'possibly' cholinergic neurons in the rat laterodorsal tegmental nucleus during sleep and wakefulness.

The cytoarchitecture of the interfascicular nucleus and ventral tegmental area of Tsai in the rat.

Intrinsic connections within the pedunculopontine tegmental nucleus are critical to the elaboration of post-ictal antinociception.