Descending projections of the posterior nucleus of the hypothalamus:Phaseolus vulgaris...

THE JOURNAL OF COMPARATIVE NEUROLOGY 374507431 (1996)

Descending Projections of the Posterior Nucleus of the Hypothalamus: PhaseoZus

vuZgaris Leucoagglutinin Analysis in the Rat

ROBERT P. VERTES AND ALISON M. CRANE Center for Complex Systems, Florida Atlantic University, Boca Raton, Florida 33431

ABSTRACT No previous report in any species has systematically examined the descending projections

of the posterior nucleus of the hypothalamus (PH). The present report describes the descending projections of the PH in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin. PH fibers mainly descend to the brainstem through two routes: dorsally, within the central tegmental tract; and ventromedially, within the mammillo-tegmental tract and its caudal extension, ventral reticulo-tegmental tracts. PH fibers were found to distribute densely to several nuclei of the brainstem. They are (from rostral to caudal) 1) lateral/ ventrolateral regions of the diencephalo-mesopontine periaqueductal gray (PAG); 2) the peripeduncular nucleus; 3) discrete nuclei of pontomesencephalic central gray (dorsal raphe nucleus, laterodorsal tegmental nucleus, and Barrington's nucleus); 4) the longitudinal extent of the central core of the mesencephalic through medullary reticular formation (RF); 5) the ventromedial medulla (nucleus gigantocellularis pars alpha, nucleus raphe magnus, and nucleus raphe pallidus); 6) the ventrolateral medulla (nucleus reticularis parvocellularis and the rostral ventrolateral medullary region); and 7) the inferior olivary nucleus. PH fibers originating from the caudal PH distribute much more heavily than those from the rostral PH to the lower brainstem.

The PH has been linked to the control of several important functions, including respiration, cardiovascular activity, locomotion, antinociception, and arousaliwakefulness. It is likely that descending PH projections, particularly those to the PAG, the pontomesencephalic RF, Barrington's nucleus, and parts of the ventromedial and ventrolateral medulla, serve a role in a PH modulation of complex behaviors involving an integration of respiratory, visceromotor, and somatomotor activity.

Indexing terms: periaqueductal gray, peripeduncular nucleus, mesopontine reticular formation,

": 1996 Wiley-Liss, Inc.

Barrington's nucleus, inferior olive

The posterior nucleus of the hypothalamus (PHI is a large nucleus that extends from the caudal end of the hypothalamus rostrally to the dorsomedial nucleus of the hypothalamus (Swanson, 1992; Vertes et al., 1995). I t is bordered medially by the third ventricle and laterally by the lateral hypothalamus. Although the anatomical connections of the hypothalamus have been fairly extensively examined, particularly those of the paraventricular and lateral nuclei of the hypothalamus (for review, see Swanson et al., 19871, few reports in any species have analyzed the ascending or descending projections of the PH.

Of the relatively few studies (Veazey et al., 1982; Hosoya, 1985; Holstege, 1987; Allen and Cechetto, 1992) that have examined descending projections from the posterior hypothalamic area, none have been exclusively devoted to the

posterior hypothalamic nucleus; that is, anterograde tracer injections have spanned the PH and adjoining sites: the PH and the supramammillary nucleus (Veazey et al., 19821, the PH and the dorsal hypothalamic area (Hosoya, 1985; Holstege, 1987), and the PH and the perifornical region (Allen and Cechetto, 1992).

The PH has been linked to the control of various functions including respiration, cardiovascular activity, locomotion, antinociception, sleep/wakefulness, and the modulation of the hippocampal electroencephalogram (EEG) (Shik and Orlovsky, 1976; Buccafusco and Brezenoff, 1979; El-

Accepted June 9,1996. Address reprint requests to Robert P. Vertes, Center for Complex Sys-

tems, Florida Atlantic University, Boca Raton, FL 33431.

O 1996 WILEY-LISS, INC.

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dridge et al., 1981, 1985; Carstens, 1982; Ohta et al., 1985; DiMicco et al., 1986; DiMicco and Abshire, 1987; Mori, 1987; Martin et al., 1988; Waldrop et al., 1988; Wible et al., 1988; Lin et al., 1989; Pare et al., 1989; Spencer et al., 1990; Bland et al., 1993,1995; Sinnamon, 1993). Recent evidence indicates that the PH may be an important site for the integration of visceromotor/somatomotor activity subserving a number of complex functions including exercise,

R.P. VERTES AND A.M. CRANE

escapeiflight, and the defense reaction (DiScala et al., 1984; Schmitt et al., 1985; Yardley and Hilton, 1986; Shekhar and DiMicco, 1987; Lammers et al., 1988; Wible et al., 1988; Vertes et al., 1995).

The PH ultimately exerts a modulatory influence on viscerosomatic activity through direct and/or indirect projections to autonomic/sensorimotor nuclei of the brainstem and spinal cord. In view of a key role for the PH in various

AP APN

BAR BC BCx BMA BP C CEM CG CL CN COA COAP CP c - P c-PH CTT cu CUN CVLM DK DMh DR ECU EN F FN FR FrC G7 IC ICP IF INC I 0 IMD IP N KF LA LC LDT LGNd LH LHY LP LPB LPl LR LTF LV LVE MB MEA MGN MH ML M05 MPB MR MRF MT MTT M v NA

A&

area postrema anterior pretectal nucleus cerebral aqueduct Barrington’s nucleus brachium conjunctivum decussation of brachium conjunctivum basomedial nucleus of amygdala brachium pontis cerebellum central medial nucleus of thalamus central gray central linear nucleus cochlear nucleus cortical nucleus of amygdala cortical nucleus of amygdala, posterior part cerebral peduncle caudate-putamen caudal part of posterior nucleus of hypothalamus central tegmental tract cuneate nucleus nucleus cuneiformis caudo-ventrolateral medullary area nucleus of Darkschewitsch dorsomedial nucleus of hypothalamus dorsal raphe nucleus external cuneate nucleus endopiriform nucleus fornix facial nucleus fasciculus retroflexus frontal cortex genu of facial nerve inferior colliculus inferior cerebellar peduncle interfascicular nucleus nucleus incertus inferior olive intermediodorsal nucleus of thalamus interpeduncular nucleus inferior vestibular nucleus Kolliker-Fuse nucleus lateral nucleus of amygdala locus coeruleus laterodorsal tegmental nucleus lateral geniculate nucleus, dorsal division lateral habenula lateral hypothalamus lateroposterior nucleus of thalamus lateral parabrachial nucleus LP of thalamus, lateral division lateral reticular nucleus lateral tegmental field (of medulla) lateral vestibular nucleus lateral ventricle mammillary bodies medial nucleus of amygdala medial geniculate nucleus medial habenula medial lemniscus motor nucleus of trigeminal nerve medial parabracbial nucleus median raphe nucleus mesencephalic reticular formation mammillothalamic tract mammillotegmental tract medial vestibular nucleus nucleus ambiguus

Abbreviations

NGC NGCa NGCv NLL NPC NTB NTS NRV N3 N6 N12 OCC PaC PAG PB PCR PF PGN PHA-L Pic p-mRF PMd PMv PN PN5 PO PP PPN PPT PrC PRH PT RM RN RO RP RPA RPC r-PH RPO RR RT RTG RVLM sc SCd SCi SN SNC SN5 so SPF ST ST5 SUM sv TeC W M VRTG VT VTA ZI 3 v 4 v 7 8

nucleus gigantocellularis nucleus gigantocellularis-pars alpha nucleus gigantocellularis-pars ventralis nucleus of lateral lemniscus nucleus of posterior commissure nucleus of trapezoid body nucleus of solitary tract nucleus reticularis ventralis oculomotor nucleus abducens nucleus hypoglossal nucleus occipital cortex parietal cortex periaqueductal gray matter parabigeminal nucleus parvocellular reticular nucleus parafascicular nucleus of thalamus nucleus paragigantocellularis (lateralis) Phaseolus uulgar~s-leucoagglutinin piriform cortex pontomedullary reticular formation premammillary nucleus, dorsal division premammillary nucleus, ventral division nucleus of pons principal nucleus of trigeminal nerve posterior nucleus of thalamus peripeduncular nucleus posterior pretectal nucleus pedunculopontine tegmental nucleus perirhinal cortex prepositus hypoglossal nucleus pyramidal tract raphe magnus red nucleus raphe obscurus raphe pontis raphe pallidus nucleus reticularis pontis caudalis rostra1 part of posterior nucleus of hypothalamus nucleus reticularis pontis oralis retrorubral area reticular nucleus of thalamus reticular tegmental nucleus of pons rostro-ventrolateral medullary area superior colliculus superior colliculus, deep layer superior colliculus, intermediate substantia nigra substantia nigra-pars compacta spinal nucleus of trigeminal nerve superior olive subparafascicular nucleus of thalamus stria terminalis spinal trigeminal tract supramammillary nucleus superior vestibular nucleus temporal cortex ventral posteromedial nucleus o f thalamus ventral reticulotegmental tracts ventral tegmental nucleus (of Gudden) ventral tegmental area zona incerta third ventricle fourth ventricle facial nerve vestibulocochlear nerve

DESCENDING PROJECTIONS OF PH 609

5-minute, DAB (same concentration) incubation to which 0.018% Ha02 had been added. Sections were then rinsed again in PBS (3 x 1 minute) and mounted onto chrome- alum gelatin-coated slides. An adjacent series of sections from each rat was stained with cresyl violet for anatomical reference.

Sections were examined using lightfield and darkfield optics. PHA-L-labeled cells (at injection sites) and fibers were plotted onto maps constructed from adjacent Nissl- stained sections (see Figs. 2, 7). The main criteria used to distinguish labeled terminals from fibers of passage were 1) the presence or essential absence of axon/terminal specializations; and 2) the degree of axonal branching. Terminal sites were typically characterized by a dense array of highly branched axons containing numerous specializations (vari- cosities, terminal boutons), whereas passing fibers exhib- ited minimal branching and contained few specializations. Representative samples of material judged particularly useful in emphasizing and/or clarifying points of text were illustrated with photomicrographs.

behaviors and the fact that no previous report has comprehensively examined the descending projections of the PH, we analyzed PH projections to the brainstem by using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin (PHA-L; Gerfen and Sawchenko, 1984).

We showed that the PH projects densely to several structures of the brainstem including (from rostral to caudal) lateral/ventrolateral regions of the diencephalo- mesopontine periaqueductal gray, the peripeduncular nucleus, Barrington’s nucleus, the longitudinal extent of the medial pontomesencephalic reticular formation, the nucleus raphe magnus, the nucleus raphe pallidus, the ventrolateral medulla (nucleus reticularis parvocellularis and the rostral ventrolateral medullary region), and the inferior olive.

MATERIALS AND METHODS Single injections of PHA-L were made into the posterior

hypothalamus of 42 male Sprague-Dawley (Charles River, Wilmington, MA) rats weighing 275-325 g. We previously described the location and boundaries of the PH for the rat (Vertes et al., 1995, Fig. 1, p. 92) (see also Swanson et al., 1987; Swanson, 1992). Single injections of PHA-L were also made into nuclei adjacent to the PH (control injections) of 17 male Sprague-Dawley rats of the same weight.

Powdered lectin from Phaseolus vulgaris leucoagglutinin was reconstituted to 2.5% in 0.05 M sodium phosphate buffer, pH 7.4. The PHA-L solution was iontophoretically deposited in the brains of anesthetized rats by means of a glass micropipette with an outside tip diameter of 40-60 pm. Positive direct current (5-10 FA) was applied through a Grass stimulator (Model 88) coupled with a high-voltage stimulator (Frederick Haer Co., Brunswick, ME) at 2 seconds “on”i2 seconds “off” intervals for 30-40 minutes. After a survival time of 7-10 days, animals were deeply anesthetized with sodium pentobarbital and perfused trans- cardially with a buffered saline wash (pH 7.4, 300 ml/ animal) followed by fixative (2.5% paraformaldehyde, 0.2- 0.5% glutaraldehyde in 0.05 M phosphate buffer, pH 7.4) (300-500 mlianimal), and then by 10% sucrose in the same phosphate buffer (150 ml/animal). The brains were re- moved and stored overnight at 4°C in 20% sucrose in the same phosphate buffer. On the following day, 50 pm frozen sections were collected in phosphate-buffered saline (PBS, 0.9% sodium chloride in 0.01 M sodium phosphate buffer, pH 7.4) and incubated for 1 hour in diluent 110% normal rabbit serum (Colorado Serum, Denver, CO) and 1% Triton X-100 (Sigma Chemicals, St. Louis, MO), in PBSI. Sections were then incubated overnight (14-17 hours) at 4°C in primary antiserum directed against PHA-L (biotinylated goat anti-PHA-L, Vector Labs, Burlingame, CA) at a dilution of 1:500 in diluent. The next day, sections were washed 5 times for 5 minutes each (5 x 5 minutes) in PBS, and then incubated in the second antiserum (rabbit anti-sheep I&, Pel-Freez, Rogers, AK) at a dilution of 1500 in diluent for 2 hours. Sections were rinsed again (5 x 5 minutes) and incubated with peroxidase-antiperoxidase (goat origin, Sternberger Monoclonals, Baltimore, MD) at a dilution of 1:250 for 2 hours. The last two incubations were repeated (double-bridge procedure), with 5 x 5 minute rinses following each incubation, for 1 hour each. After 5 x 5 minute rinses, the sections were incubated in 0.05% 3,3’diaminoben- zidine (DAB) in PBS for 10 minutes, followed by a second,

RESULTS Two of 42 cases with injections in the posterior nucleus of

the hypothalamus are described in detail: one with an injection in the caudal part of PH (case 504) and the other with an injection in the rostral part of PH (case 536). The injections were restricted to the respective parts (rostral or caudal) of PH. In our previous analysis of the ascending projections of PH, we included a cytoarchitectonic map of PH (Vertes et al., 1995, Fig. 1, p. 92). Briefly, as shown in that figure, the PH extends from the level of the mammillary body forward to the dorsomedial nucleus of the hypothalamus. At caudal levels, the PH is bordered laterally by the ventral tegmental nucleus, dorsally by the periaqueductal gray, and ventrally by the supramammillary nucleus. At rostral levels, the PH is bordered ventrally by the dorsal premammillary nucleus, dorsally by the central medial nucleus of the thalamus, laterally by the lateral hypothala- musizona incerta, and medially by the third ventricle.

The labeling shown with the two illustrated cases is representative of that seen with other rostral or caudal PH cases that have not been illustrated. Material from 17 nonschematically illustrated control cases (injections in nuclei adjacent to PH) is also presented and discussed. Figure 1 depicts injection sites for the schematically illustrated caudal (Fig. lA,A‘) and rostral (Fig. lB,B‘) PH cases as well as for control cases with injections in the dorsomedial (Fig. lC,C’) and lateral (Fig. lD,D’) nuclei of the hypothalamus. The areas outlined by arrows in the Nissl- stained sections (Fig. lA‘,B‘,C’,D‘) represent the boundaries of respective nuclei at the level of injection, i.e., not the area of the injections. As shown, each of the injections (Fig. IA-D) is confined to the boundaries of the respective nuclei.

Posterior nucleus of the hypothalamus-caudal part (case 504)

Figure 2 schematically depicts the distribution of labeled fibers in the brainstem produced by a PHA-L injection in the caudal part of PH. Labeled fibers from the site of injection coursed caudally to the brainstem mainly via two routes: dorsally, through the central tegmental tract (CTT) (lateral to the periaqueductal gray), and ventromedially,

Figure 1

DESCENDING PROJECTIONS OF PH

through the mammillotegmental tract (MTT). Labeled fibers distributed over a wide cross-sectional area of the rostral midbrain (Fig. 2B,C). Moderately to densely labeled sites were the anterior and medial pretectal nuclei, the central tegmental field of the midbrain [or mesencephalic reticular formation (MRF)], the nucleus of the posterior commissure, the substantia nigra-pars compacta (SNC), the peripeduncular nucleus (PP), the intermediate layer of the superior colliculus, and lateral parts of the periaqueductal gray (PAG) (Fig. 2B,C). The lateral PAG was densely labeled ipsilaterally and moderately labeled contralaterally. Overall, labeling was considerably stronger ipsilaterally than contralaterally. There was essentially an absence of labeling within the red nucleus (Fig. 2B,C).

Labeled axons continued to mainly descend within the CTT, dorsally, and ventral reticulotegmental tracts (VRTG), ventrally, farther caudally in the brainstem (Fig. 2D,E). At mid-levels of the midbrain (Fig. 2D), labeled fibers spread widely throughout the dorsal and ventral tegmentum, distributing to lower and intermediate layers of SC (SCi and SCd), the lateraliventrolateral PAG, the MRF, the PP (as well as the region dorsal to PP), and to the retrorubral nucleus (RR) (Fig. 2D). The dorsal raphe nucleus was lightly labeled. The photomicrograph of Figure 3A shows labeling of the peripeduncular nucleus, dorsal to the substantia nigra.

At the rostral pons (Fig. 2E,F), the dorsal tract (CTT) was no longer recognizable as a distinct bundle; labeled fibers continued to descend through the ventral bundle (VRTG), located medialidorsomedial to the medial lemniscus (Fig. 2E-G) and farther caudally, dorsal to the trapezoid body (Fig. 2H,I). The nucleus pontis oralis (RPO) and lateral/ventrolateral regions of the pontine central gray (CG) were densely labeled; the laterodorsal tegmental (LTD) and pedunculopontine tegmental (PPT) nuclei were moderately labeled; and the nucleus cuneiformis, as well as dorsal and median raphe nuclei, was lightly labeled (Fig. 2E,F).

Further caudally in the pons, labeled fibers continued to occupy a relatively widespread area of the pontine tegmentum, distributing densely to nucleus pontis caudalis and to Barrington’s nucleus (of the pontine CG) as well as lightly to moderately to LTD, the medial parabrachial nucleus (area), the nucleus tegmenti pontis, and the raphe pontis (Fig. 2G,H). Few, if any, labeled axons were present in the locus coeruleus (LC). The photomontage of Figure 4 shows dense labeling of Barrington’s nucleus (BAR) and nucleus pontis caudalis; note the essential absence of labeling in LC, lateral to BAR.

At the rostral medulla (Fig. 2I-K), relatively heavy labeling was observed: 1) along the midline, mainly ventrally within nucleus raphe magnus and raphe pallidus; and 2) immediately lateral to the midline within nucleus gigan-

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tocellularis (NGC), and nucleus gigantocellularis-pars alpha (NGCa), ventral to NGC. The lateral parvocellular reticular nucleus (PCR) was moderately labeled. At these same levels (Fig. 2J,K), a well-defined group of labeled axons coursed dorsally around the facial nucleus to distribute to the retrofacial area, rostrally (Fig. 51, and to the rostral ventrolateral medullary region (RVLM), caudally (Fig. 2L,M). With the exception of a minor distribution to the facial nucleus (FN), very few labeled axons terminated within any cranial sensorimotor nuclei of the medulla.

A principal target of labeled axons at mid- to lower levels of the medulla (Fig. 2M,N) was the inferior olive (10). Labeled fibers distributed throughout the rostro-caudal extent of the ipsilateral I 0 and to each of its major subdivisions: the principal, medial accessory, and dorsal accessory olivary nuclei (Fig. 6). Additional terminal sites were NGC, NGC-pars ventralis, PCR, and RVLM as well as raphe obscurus (RO) and raphe pallidus (RPA) (Fig. 6). At the caudal medulla (Fig. 2N,O), labeled fibers distributed heavily to 10; moderately to nucleus reticularis dorsalis, reticularis ventralis, PCR, and caudal ventrolateral medulla (CVLM); and lightly to nucleus paragigantocellularis lateralis, RO, and RPA.

Fig. 1. Low-magnification lightfield photomicrographs of Phaseolus uulguris-leucoagglutinin (PHA-L) immunostained and corresponding cresyl violet-stained sections showing the site of injections in the caudal part of the posterior nucleus of the hypothalamus (PH) (A,A’), the rostral part of PH (B,B‘), the dorsomedial nucleus of the hypothalamus (C,C’), and the caudal pole of the lateral hypothalamus (D,D‘). The regions demarcated by arrows in A’,B‘,C‘,D’ represent the hound- a rks of respective nuclei (caudal and rostral PH, dorsomedial and lateral hypothalamus) at the levels of the injection. Note that each of the injections (A-D) is confined to the boundaries of the respective nuclei. Scale bar = 500 pm.

Posterior nucleus of the hypothalamus-rostra1 part (case 536)

Figure 7 schematically depicts the distribution of labeled fibers in the brainstem produced by a PHA-L injection in the rostral part of PH. Labeled axons from the site of injection mainly descended dorsally within the CTT (and the region ventraliventrolateral to it) and only in a minor way through ventral routes [i.e., VRTG]. Similar to the caudal PH case, labeled fibers distributed over a widespread region of the midbrain (Fig. 7B,C). Principal sites of termination were lateraliventromedial parts of the midbrain PAG (Fig. 7B,C), the entire expanse of the central tegmental field (Fig. 7B,C), and the PP (Fig. 7C). The photomicrograph of Figure 8A depicts pronounced labeling of the lateral PAG at approximately the level shown schematically in Figure 7C. Labeled fibers distributed moderately to the dorsomedial PAG, to the nucleus of the posterior commissure, and to central linear nucleus (CL), and lightly to the intermediate and deep layers of SC, to the anterior pretectal nucleus, and to SNC. Apart from the contralateral PAG, which was moderately labeled, few labeled fibers were present contralateral to the injection at these levels (Fig. 7B,C) or further caudally throughout the brainstem.

At the caudal midbrain (Fig. 7D,E), labeled fibers continued to distribute heavily to the midbrain PAG and to the laterally adjacent tegmentum (Fig. 8B), dorsally, and to the MRF and PP, ventrally. Deep layers of SC (SCi, SCd) and the SNC were lightly labeled.

The primary targets of labeled fibers at the rostral pons (Fig. 7F,G) were lateral regions of the PAG (Fig. 8C,D), the dorsal raphe nucleus, LDT, and nucleus pontis oralis. The photomontage of Figure 9 shows labeled fibers spread throughout RPO. Lightly to moderately innervated sites were the SCi, SCd, PPT, the retrorubral nucleus, nucleus cuneiformis, and the lateral reticular tegmental field of the pons.

There was marked and progressive decline in the inten- sity of labeling beginning approximately at the level of the mid-pons and continuing through to the caudal end of the

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Fig. 2. A-0: Schematic representations of the labeling present in selected sections through the brainstem produced by a PHA-L injection (dots in A) in the caudal part of the posterior nucleus of the hypothalamus (case 504).

613 DESCENDING PROJECTIONS OF PH

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Figure 2 (Continued.)

brainstem. At the caudal pons (Fig. 7H,I), labeled fibers distributed relatively heavily to Barrington’s nucleus (Fig. 3B), moderately to RPC and to LDT, and lightly to PPT, the

medial and lateral parabrachial nuclei, and raphe pontis. The locus coeruleus was at best sparsely labeled. The small numbers of labeled fibers present at the level of the rostra1

614 R.P. VERTES AND A.M. CRANE

Fig. 3. A: Darkfield photomicrograph of a transverse section through the midbrain showing labeling in the peripeduncular nucleus (PP), dorso-dorsolateral to the substantia nigra (SN) produced by the injec-

tion of case 504. B: Darkfield photomicrograph of a transverse section through the pons showing labeling in Barrington’s nucleus produced by the injection of case 536. Scale bar = 500 pm.

medulla spread relatively homogeneously throughout the medullary tegmentum terminating moderately in rostral NGC, and lightly in the caudal NGC, NGCa, PCR, and nucleus raphe magnus (Fig. 7J,K). Only scattered labeling

was observed at mid- to caudal levels of the medulla (Fig. 7L-N), essentially restricted to NGC, nucleus reticularis ventralis, and to the lateral tegmental field of the lower medulla.


Fig. 4. Darkfield photomicrograph of a transverse section through the pons showing labeling throughout the nucleus pontis caudalis (RPC) and dorsally within Barrington’s nucleus. Labeling produced by the injection of case 504. Scale bar = 500 pm.

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Fig. 5. Darkfield photomicrograph of a transverse section through the medulla showing labeling ventromedially, within the nucleus gigantocellularis-pars alpha, dorsal to the pyramidal tract (PT); and

ventrolaterally within the ventrolateral medullary tegmentum, lateral to the facial nucleus (FN). Labeling produced by the injection of case 504. Scale bar = 500 km.

Other PH injections As described above, there were significant differences in

the brainstem distribution of labeled fibers from caudal and rostra1 PH. By contrast, there were only minor differences in patterns of brainstem labeling produced by injections in the medial vs. lateral or dorsal vs. ventral PH.

Control injections-structures bordering the PH

midline. The former included the ventromedial PAG, DR, LDT, LC, and nucleus incertus; the latter included the

linear nucleus, the median raphe, and the rostrd pole of raphe magnus, The two the appearance of a T-shaped pattern of labeling. The lateral and medial parabrachial nuclei were lightly labeled.

Injections in SUM gave rise to labeled fibers virtually entirely targeted for the ventroiventromedial PAG and the DR (see also Vertes. 1992).

of labeling interseded,

Control injections were made in several structures bordering PH including the ventral tegmental area (VTA), the supramammillary nucleus (SUM), and the dorsal premammillary nucleus (PMd), ventrally (or postero-ventrally); the dorsomedial nucleus (DMh) of the hypothalamus, rostrally; and the lateral hypothalamus (LHy), laterally. With the exception of injections in the lateral hypothalamus, injections in each of these sites produced light-to-moderate labeling within the brainstem, largely confined to the midbrain and the pons.

Injections in VTA produced relatively moderate labeling mainly restricted to nuclei of the mesopontine gray and the

Injections of the dorsal premammillary nucleus produced labeling largely restricted to the mesopontine gray and immediately surrounding regions. Labeled PMd fibers distributed throughout the longitudinal extent of the dorsal/ dorsolateral CG from the caudal diencephalon (periventricular gray) to the caudal pons. PMd fibers terminating within the lateral PAG formed two distinct groups: one at the 8:OO-9:00 position and the other at the 11:OO-12:OO position. Labeled PMd axons also terminated moderately within the medial pretectal area, dorsal to the posterior commissure, the mesopontine lateral tegmental field, lateral to PAG, the DR, and dorsal parts of LC.


Fig. 6. Darkfield photomicrograph of a transverse section through the caudal medulla showing labeling along the midline within nucleus raphe obscurus and raphe pallidus and ventrolaterally within the

rostral ventrolateral medullary region (RVLM), as well as dense labeling within the inferior olivary nucleus (10). Labeling produced by the injection of case 504. Scale bar = 500 pm.

Injections in the dorsomedial nucleus of the hypothalamus just rostral to PH (Fig. lC,C’) gave rise to a pattern of labeling similar to that seen with PMd injections. DMh injections resulted in dense labeling throughout longitudinal extent of the lateral mesopontine PAG. This is depicted at one level (midbrain PAG) in the photomicrograph of Figure 10A. Additional lightly to moderately labeled sites were the dorsolateral pontine tegmentum, including PPT and the lateral parabrachial nucleus, DR, MR, LDT and LC. Labeling decreased significantly caudal to the pons.

Injections within the lateral hypothalamus (Fig. lD,D’) resulted in stronger and more widespread brainstem labeling than that seen with injections in other sites bordering PH. LHy injections produced the following pattern of labeling: 1) In the midbrain, labeled fibers distributed moderately to densely to deep and intermediate layers of the superior colliculus, the entire ipsilateral half of the PAG (labeling was heaviest mid-laterally, directly lateral to the aqueduct), the lateral mesencephalic RF, the PP, and the SNC; 2 ) in the pons, labeled fibers terminated moderately to densely in the pontine CG, DR, PPT, medial and lateral parabrachial nuclei, and the lateral/dorsolateral pontine RF; and 3) in the medulla, labeled axons distributed lightly to moderately to the lateral tegmental field (parvocellular RF), NGCa and NGC-pars ventralis, RVLM, CVLM, and

the nucleus of the solitary tract. The photomicrograph of Figure 10B depicts labeling of the lateral parabrachial nucleus and the medially adjoining rostral LC produced by the LHy injection of Figure 1D. In general, labeled LHy fibers distributed more laterally in the brainstem than did those from PH (see also Discussion).

DISCUSSION In the following account we highlight our results, com-

pare them with those of previous studies, and discuss functional implications.

The principal findings were that PH fibers distribute significantly to several structures throughout the brainstem. The main sites of termination were (from rostral to caudal) 1) lateral/ventrolateral regions of the diencephalo- mesopontine PAG; 2 ) the PP; 3) discrete nuclei of pontomesencephalic gray-the dorsal raphe nucleus, laterodorsal tegmental nucleus, and BAR; 4) the longitudinal extent of the central core of the reticular formation; 5) the ventromedial medulla (NGCa, nuclei raphe magnus and raphe pallidus); 6) the ventrolateral medulla (nucleus reticularisparvo- cellularis and RVLM); and 7) the I 0 (see Table 1; Fig. 11).

In addition, PH fibers were found to distribute moderately to the VTA, SCi and SCd, nucleus of the posterior

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Fig. 7. A-N Schematic representations of the labeling present in selected sections through the brainstem produced by a PHA-L injection (dots in A) in the rostra1 part of the posterior nucleus of the hypothalamus (case 536).


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Figure 7 (Continued.)


Fig. 8. Darkfield photomicrographs of transverse sections through the caudal diencephalon and brainstem showing labeling within the lateral periaqueductal gray at the diencephalon (A), the midbrain (B), the rostral pons (0, and the caudal pons (D). Labeling produced by the injection of case 536. Scale bar = 500 bm.

commissure, the retrorubral nucleus, and the PPT; and lightly to the SNC, caudal linear nucleus, anterior and medial pretectal nuclei, median raphe nucleus, nucleus cuneiformis, locus coeruleus, medial and lateral parabrachial nuclei, and nucleus paragigantocellularis (see Table 1; Fig. 11).

Comparisons of projections between caudal and rostral parts of PH

The major difference between projections from the caudal and rostral PH is that caudal PH fibers distribute much more heavily than do those of the rostral PH to the lower brainstem. Rostral PH injections produced only scattered labeling caudal to the exit level of the facial nerve. In contrast, significant numbers of fibers could be traced from the caudal PH to several nuclei of the lower brainstem. No major differences were observed in patterns of projections from the caudal or rostral PH to the pons and midbrain. Rostral PH fibers predominantly descended to the brain-

stem dorsally through the CTT, whereas those from the caudal PH descended via two routes, the CTT and ventral pathways (the mammillotegmental tract, rostrally, and ventral reticulotegmental tracts, caudally). Caudal PH f i - bers distributing to the lower brainstem appeared to mainly descend via the ventral tracts. There were no marked differences in projections from various parts of caudal or rostral PH, that is, medial vs. lateral or dorsal vs. ventral regions of the caudal or rostral PH.

PH projections to the brainstem: Comparisons with previous anterograde tracing studies

No previous report has comprehensively analyzed the descending projections of PH. A few studies, however, have examined descending projections from regions of the caudal hypothalamus that included parts of PH, as described below.

Veazey et al. (19821, using autoradiographic methods in the monkey, described ascending and descending projec-


Fig. 9. Darkfield photomicrograph of a transverse section through the pons showing labeling distributed throughout the nucleus pontis oralis (RPO) produced by the injection of case 536. Scale bar = 500 km.

tions from several nuclei of the caudal hypothalamus including PH. The single PH injection that was illustrated (Veazey et al., 1982) included PH and the ventrally located supramammillary nucleus. Veazey et al. (1982) described relatively substantial PH area projections to the brainstem. In general accord with present findings, they showed that

PH fibers mainly descend via the CTT and distribute heavily to the PAG, the medial RF (midbrain through rostral medulla), and several raphe nuclei including the dorsal, median, and what they termed the ventral raphe, which appears to encompass raphe magnus and the rostral part of raphe pallidus. Veazey et al. (1982) also demon-


Fig. 10. A: Darkfield photomicrograph of a transverse section through the midbrain showing labeling in the dorsolateral quandrant of the periaqueductal gray produced by a control injection in the dorsomedial nucleus of the hypothalamus. B: Darkfield photomicrograph of a

transverse section through the rostra1 pons showing labeling within the dorsolateral pons in the locus coeruleus (LC) and the peri-LC area, medially, and in the lateral parabrachial nucleus, laterally, produced by a control injection in the lateral hypothalamus. Scale bar = 500 pm.


midline hypothalamic region that appeared to include the rostral part of PH and the DMh. In accord with present findings, Hosoya (1985) demonstrated pronounced projections from PH/DMh to raphe magnus and pallidus as well as to the lateral medullary tegmental field, i.e., to the parvocellular RF, dorsally, and the RVLM region, ventrally. Hosoya (1985) also described significant PH area projections to the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus (DMV) and the area postrema (AP). This contrasts with our findings of an essential absence of PH projections to each of these sites. It is possible that the projections of Hosoya (1985) to NTS, DMV, and AP originated mainly from the DMh area and not PH.

Consistent with present findings, but partially at odds with those of Hosoya (1985), Holstege (1987) showed that tritiated amino acid injections that included the rostral part of PH produced relatively pronounced labeling of the PAG, medial RF (midbrain through upper medulla), and caudal raphe groups (magnus and pallidus), but essentially no labeling of the solitary complex, AP, or the dorsal motor vagal nucleus.

Allen and Cechetto (1992), using PHA-L or WGA-HRP, recently described projections to the brainstem from a cardiovascular pressor site located medially adjacent to the fornix which they termed the perifornical area (PFA). The caudal part of PFA overlaps with lateral aspects of PH (see their Fig. 11, p. 326). In accord with present findings, Allen and Cechetto (1992) demonstrated pronounced PFA projections to the lateral PAG and to BAR, but unlike our results, showed few PFA projections to other nuclei of the pons and midbrain and few to the medulla. These differences may involve the fact that their PFA sites only included lateral aspects of PH.

Major brainstem sites of innervation: Comparisons with previous studies and

functional implications Periaqueductal gray /dorsal tegmentum lateral to PAG.

The present results show that rostral as well as caudal PH fibers project densely to PAG; the PAG was the most densely innervated site in the brainstem. Several reports in various species have examined afferent projections to the PAG using retrograde tracers (Grofova et al., 1978; Beitz, 1982; Mantyh, 1982; Marchand and Hagino, 1983; Bandler and McCulloch, 1984; Meller and Dennis, 1986; Beart et al., 1990; Veening et al., 1991) and have shown that 1) afferents to PAG arise from widespread regions of the brain; 2) the hypothalamus is the major source of afferents to PAG; and 3) most nuclei of the hypothalamus project to PAG. With respect to PH, some studies have reported light-to- moderate PH projections to PAG (Grofova et al., 1978; Marchand and Hagino, 1983; Bandler and McCulloch, 19841, whereas others have demonstrated substantial PH projections to PAG (Beitz, 1982; Mantyh, 1982; Meller and Dennis, 1986; Beart et al., 1990). Included among the latter, Beitz (1982) showed significant numbers of retrogradely labeled cells in the PH area, primarily localized to the lateral PH Gust medial to the mammillothalamic tract), following HRP injections in the PAG of the rat. Similar results were described in the monkey (Mantyh, 1982) as well as in a subsequent report in the rat (Meller and Dennis, 1986). Beart et al. (1990) recently examined excitatory amino acid ( E M ) projections to PAG, using the retrograde transport of D[3H]aspartate in the rat, and, consistent with earlier work, showed that PH, along with the ventromedial

TABLE 1. Density of Labeling in Nuclei of the Brainstem Produced by PHA-L Injections in Rostra1 (r-PH) or Caudal (c-PH) Parts of the Posterior

Nucleus of the Hypothalamus’

Injection sites

Nuclei r-PH c-PH Ahducens n - - Anterior pretectal n. + ++ Barrington’s n. +++ +++ Caudo-ventrolateral medulla (CVLM) - + Central linear n. + + Cuneate n. - - Cuneiform n. + + Dorsal cochlear n. - - Dorsal motor n. vagus - - Dorsal raphe n ++ + Dorsal tegmental n. + + Facial n. - + Hypoglossal n. - -

Inferior wlliculus - - Inferior olive +++ Inferior vestibular n. - -

Interfascicular n. - - Interpeduncular n. - - Kolliker-Fuse n. + + Lateral reticular n. - + Lateral parabrachial n. + + Lateral vestibular n. - - Locus coeruleus + + Medial parabrachial n. + ++ Medial vestibular n. - - Median raphe n. + + Mesencephalic reticular formation +++ +++ Motor trigeminal n. - - N. amhiguus - - N. incertus + +

-

Laterodorsal tegmental n. ++ ++

N. gigantocellularis ++ +++ N. gigantocellularis, pars alpha + ++ N. gigantocellularis, pars ventralis - + N. lateral lemniscus - - N. paragigantocellularis (lateralis) - + N. pons - - N. pontis caudalis +++ +++ N. pontis oralis +++ +++ N. posterior commissure ++ + N. raphe magnus + +++ N. raphe ohscurus ++ N. raphe pallidus - +++ N raphe pontis + ++ N. reticularis dorsalis - + N. reticularis ventralis ++ N. solitaly tract - - N. trapezoid body -

Parvocellular reticular n. + ++ Pedunculopontine tegmental n. + + Prepositus hypoglossal n - -

-

-

Periaqueductal gray, diencephalon +++ +++ Periaqueductal gray, midbrain +++ +++ Periaqueductal gray. pons +++ +++ Peripeduncular n. +++ +++ Posterior pretectal n. + ++ Principal trigeminal n. - - Red n Reticular tegmental n. pons - Retroruhral area ++ ++ Rostro-ventrolateral medulla (RVLM) +++ Spinal trigeminal n. - - Substantia nigra, pars compacta + ++ Suhstantia nigra, pars reticulata - Superior colliculus + ++ Superior olive - - Superior vestibular n. - - Ventral cochlear n. - Ventral tegmental n. + -

I + , light labeling; + +, moderate labeling, + + +, dense labeling; - , absence of labeling; n , nucleus.

- - -

-

-

strated relatively pronounced PH projections to the laterodorsal and pedunculopontine tegmental nuclei and to a pontine site which, although not identified as such, appears to correspond to Barrington’s nucleus. PH projections to the lower medulla (i.e., below the level of the abducens nucleus) were not examined.

Using a combination of tracers in the rat, Hosoya (1985) examined projections to the lower brainstem from a dorsal


C-PH n l I r-PH 0 ventromedial parts of the PAG including the dorsal raphe nucleus. The notion has been put forth that PAG-elicited analgesia may be part of a global response to threatening --.. ’dv 0

MIDBRAIN APN CUN MRF NPC PA G PP PPN RR sc SNC

PONS BAR DR LC LDT LPB KF MPB MA PAG PPT RP RPC RPO

MEDULLA CVLM 10 NGC NGCa NGCv NRV PCR PGN RM RO RPA RVLM

\

MIDBRAIN APN CUN MRF NPC PAG PP PPN RR sc SNC

PONS BAR DR LC LDT LPB KF MPB MR PAG PPT RP RPC RPO

M E W LLA CVLM 10 NGC NGCa NGCv NRV PCR PGN RM RO R PA RVLM

Fig. 11. Summary diagram depicting relative densities of labeling in structures of the midbrain, pons, and medulla produced by injections in caudal (c-PH) and rostral (r-PH) parts of the posterior nucleus of the hypothalamus. Large, medium, and small arrows indicate dense, moderate, and light labeling, respectively. No arrows indicate an essential absence of labeling.

nucleus of the hypothalamus, was a rich source of hypothalamic EAA afferents to the PAG.

The PAG has been implicated in several functions including cardiorespiratory activity, vocalization, rage and aggres- sion, lordosis, and most prominently the “defense reaction” and antinociception (for review, see Depaulis and Bandler, 1991). Beginning with the early work of Abrahams and colleagues (Abrahams et al., 1960, 19641, several studies have shown that electrical or chemical activation of discrete regions of the PAG, particularly the lateral PAG, evokes a stereotypical pattern of defense-associated behaviors (Jur- gens and Pratt, 1979; Bandler et al., 1985; Depaulis and Vergnes, 1986; Hilton and Redfern, 1986; Yardley and Hilton, 1986; Carrive et al., 1987, 1989; Bandler and Carrive, 1988; Jurgens, 1994).

It is also well established in several species that stimulation of the PAG produces analgesia (antinociception) (Rey- nolds, 1969; Mayer et al., 1971; Giesler and Liebeskind, 1976; Fardin et al., 1984a,b). PAG sites involved in antinociception include lateral regions of the PAG (i.e., overlap- ping with sites involved in the defense reaction) and

situations (Besson et al., 1991; Lohck, 1991). In this regard, Lovick (1991) stated: “Under the extreme or emer- gency situations which evoke attack or defensive behavior, an acute period of analgesia may have considerable survival value. During the analgesic phase the normal protective reflexes to a noxious stimulus (e.g., flexor withdrawal. immobilization of the affected part) would be suppressed. thus giving priority to the execution of movements directed toward the survival of the whole animal.”

Peripeduncular nucleus. We demonstrated pronounced projections from the rostral as well as caudal PH to the PP of the lateral midbrain tegmentum. To our knowledge, only a single report in the rat (Amault and Roger, 1987) has systematically examined the connections of PP. They showed that PP afferents mainly originate from auditory and visual structures (inferior and superior colliculi, nucleus of lateral lemniscus, and secondary auditory cortex), parts of the brainstem (PAG, cuneiform nucleus), and the hypothalamus. The primary sources of hypothalamic projections to PP were the ventromedial nucleus of the hypothalamus (VM) and PH. The PP has been strongly implicated in the lordosis reflex (Hansen and Gummesson, 1982; Lopez and Carrer, 1982, 1985). The well-documented modulatory role of the VM on lordosis (Pfaff and Sakuma, 1979a,b) may in part be mediated through projections to PP. The PH projections to PP may serve a complementary function, that is, a facilitation or enhancement of lordosis.

Mesencephalic reticular formationldorsolateral mesopontine tegmentum. We showed that caudal as well as rostral PH fibers distribute 1) massively throughout the central midbrain tegmentum (mesencephalic reticular formation) and 2) lightly to moderately to cell groups of the dorsolateral mesopontine tegmentum including nucleus cuneiformis, the PPT, and the parabrachial complex (medial and lateral parabrachial nuclei, Kolliker-Fuse nucleus ). As described below, these findings are generally in accord with those of previous reports.

Although an early report in the rat (Shammah-Lagnado et al., 1983) demonstrated light retrograde cell labeling in the PH following horseradish peroxidase (HRP) injections in the MRF, other reports in cats (Steriade et al., 1982; Bayev et al., 1988; Pare et al., 19891, also using retrograde tracers, have described pronounced PH projections to the MRF.

A number of recent analyses of afferents to the nuclei of the dorsolateral mesopontine tegmentum have, in accord with present results, described relatively small numbers of retrogradely labeled cells in PH following retrograde tracer injections in the PPT (Semba and Fibiger, 1992; Steininger et al., 19921, the medially adjacent (to PPT) extrapyramidal area (MEA) (Steininger et al., 19921, or the parabrachial complex (Moga et al., 1990). In contrast, the lateral hypothalamus was shown to project heavily to each of these sites (Moga et al., 1990; Semba and Fibiger, 1992; Steininger et al., 1992).

A possible role in wakefulness1 arousal. Based on early observations of von Economo (1926) on patients with encephalitis lethargica and on later work in cats (Nauta, 1946; Swett and Hobson, 1968) that showed that PH area damage/lesions produced a profound and enduring state of somnolence, the PH came to be regarded as a “waking center.” The “waking center” was

PH-MRF projections:


thought to reciprocally interact with a “sleep center” of the basal forebrain (McGinty and Sterman, 1968; Szymusiak and McGinty, 1986) in the control of sleepiwaking states (for review, see Steriade and McCarley, 1990; Vertes, 1990). A role for PH in arousaliwakefulness has recently been strengthened by the demonstration that 1) PH cells, including those projecting to the MRF, discharge regularly and at significantly higher rates in waking than in slow-wave sleep (SWS) (Pare et al., 1989; Sakai et al., 1990), and 2) injections of the GABA agonist, muscimol, into PH, which suppresses the activity of PH cells, produced a long-lasting hypersomnia (persistence of deep SWS) (Lin et al., 1989).

It is well documented that the mesencephalic reticular formation serves a critical role in cortical EEG activation/ arousal (for review, see Vertes, 1984, 1990; Steriade and McCarley, 1990). Several recent studies (El Mansari et al., 1989; Steriade et al., 1990; Kayama et al., 1992) have demonstrated a complementary role for the PPT in these processes: that is, a cholinergic PPT modulation of cortical EEGibehavioral arousal. The PH to MRFiPPT projections demonstrated here and previously (Steriade et al., 1982; Bayev et al., 1988; Pare et al., 1989; Semba and Fibiger, 1992; Steininger et al., 1992) may provide an excitatory drive to the MRFiPPT in the control of cortical activation of sleep and waking. In this regard, Steriade and McCarley (1990) recently stated that PH to MRF projections may serve as “a possible source of tonic impingement on midbrain reticular neurons involved in the maintenance of thalamocortical activation processes.”

PH-dorsolateral mesopontine projections: A possible role in locomotion. Stimulation of a region of the caudal hypothalamus in and around PH produces locomotion; this region has been designated the “subthalamic locomotor region” (SLR) (Shik and Orlovsky, 1976; Mori, 1987; Armstrong, 1988; Sinnamon, 1993). The SLR (as well as other forebrain systems controlling locomotion) appears to mainly act through locomotor networks of the brainstem including the mesencephalic locomotor region (MLR) of the dorsolateral mesopontine tegmentum and the medial pontomedullary RF (Swanson et al., 1984; Mori, 1987; Bayev et al., 1988; Garcia-Rill and Skinner, 1988; Levy and Sinna- mon, 1990; Sinnamon, 1993). Few reports have examined connections between the SLR and MLR. In accord with present findings, Garcia-Rill et al. (1983) described relatively light projections from the “SLR area” to the MLR. Similarly, Bayev et al. (1988) reported the presence of only “a small number” of retrogradely labeled cells in the SLR region following HRP injections in the MLR. These findings suggest that SLR (or PH area) does not exert a significant influence on the MLR in the control of locomotion.

Medial pontomedullary reticular formation (nucleus pontis oralis, nucleus pontis caudalis, nucleus gigantocellularis). We showed that fibers from the rostral and caudal PH heavily innervate the central core of the pontine reticular formation (RPO and RPC). Caudal PH fibers distribute densely, and rostral PH fibers moderately, to the medial medullary RF (nucleus gigantocellularis). In contrast to present findings, Shammah-Lagnado et al. (19871, using retrograde techniques, described light PH projections to the medial pontine RF. Bayev et al. (19881, on the other hand, demonstrated significant numbers of retrogradely labeled cells in PH following HRP injections in the medial medullary RF, and, in accord with present data, showed greater numbers of labeled cells in the caudal than rostra] PH.

625

PH-pontomedullary RF projections: A possible role in locomotion. A relatively extensive longitudinal zone of the medial pontomedullary RF regulates locomotion presum- ably through direct action on spinal locomotor networks. I t has been shown that 1) electrical or chemical activation of the medial pontomedullary RF (p-mRF) elicits locomotion (Mori et al., 1978; Drew and Rossignol, 1984; Ross and Sinnamon, 1984; Garcia-Rill and Skinner, 1987; Noga et al., 1988); 2) cooling or electrolytic lesions of the p-mRF reversibly or irreversibly abolish spontaneous or MLR- induced locomotion (Shefchyk et al., 1984; Jell et al., 1985; Noga et al., 1991); and 3) the discharge of a large percentage of units of the p-mRF is correlated with locomotion in the decerebrate or intact preparation (Shimamura et al., 1982, 1985; Drew et al., 1986; Perreault et al., 1993). The strong PH to p-mRF projections demonstrated here and previously (Bayev et al., 1988) would suggest a relatively pronounced influence of the subthalamic locomotor region on pontomedullary RF locomotor systems.

PH-pontomedullary projections: A possible role in the control of the hippocampal EEG. The pontine RF, particularly nucleus pontis oralis of the rostral pons, serves a well-documented role in the control of the theta rhythm of the hippocampus (Vertes, 1981, 1986; Nunez et al., 1991; Bland and Colom, 1993; Vertes et al., 1993). It has recently been shown that a subset of PH cells discharges tonically with the hippocampal theta rhythm (Bland et al., 1993, 1995) and that pharmacological blockade of the PH area blocks spontaneous as well as RPO-elicited theta (Kirk and McNaughton, 1993; Oddie et al., 1994). It has been proposed that the caudal diencephalon (supramammillary nucleus and PH) serves as an important relay between RPO and the septum-hippocampus in the generation of hippocampal theta (Vertes, 1986; Bland and Colom, 1993; Kirk and McNaughton, 1993; Kocsis and Vertes, 1994; Oddie et al., 1994). The present demonstration of substantial PH projections to RPO suggests a descending (or reciprocal) (Vertes and Martin, 1988) PH influence on RPO, possibly involved in the control of the theta rhythm.

We showed that fibers of the caudal as well as rostral PH terminate densely in Barring- ton’s nucleus (BAR). A recent study in the rat described widespread afferents to BAR (Valentino et al., 1994) including pronounced projections from a region of the posteromedial hypothalamus medially adjacent to the fornix, designated the “perifornical region.” The perifornical region overlaps with PH. Allen and Cechetto (1992) previously demonstrated dense projections from essentially this same “perifornical” site to BAR.

BAR (Barrington, 1921) serves a well-documented role in micturition (Loewy et al., 1979; Holstege et al., 1986; Sugaya et al., 1987; Noto et al., 1988). The pronounced PH/PH area projections to BAR shown here and previously (Allen and Cechetto, 1992; Valentino et al., 1994) would suggest a PH modulation of micturition, possibly as part of a constellation of PH-controlled events subserving motivational/emotional behavior.

Ventromedial medulla [nucleus raphe magnus (RM), RPA, NGCa]. We showed that fibers of the caudal PH distribute moderately densely to RM and RPA and lightly to moderately to NGCa, whereas those of the rostral PH project lightly to all three sites.

Several reports have shown that PH is a source of pronounced projections to RPA (Veazey et al., 1982; Pe-

Barrington’s nucleus.

626

schanski and Besson, 1984; Hosoya, 1985; Hosoya et al., 1987; Luppi et al., 1987; Holstege, 1987) and significant but less dense projections to RM. An early study by Veazey et al. (1982) demonstrated pronounced PH area projections in monkeys to the “ventral raphe,” which appeared to encompass RM and RPA. In an initial report, Hosoya (1985) demonstrated that fibers originating from a region of the dorsomedial hypothalamus that overlaps with PH distribute widely throughout the ventromedial medullary tegmentum to RM, RPA, and NGCa, and subsequently showed that wheat germ agglutinin (WGA)-HRP injections in RPA produced dense cell labeling in this dorsomedial hypothalamic zone (Hosoya et al., 1987). This latter finding is consistent with a report by Luppi et al. (1987) showing massive cell labeling in PH following retrograde tracer injections in RPA.

The PH projection to raphe magnus is less pronounced than that to RPA. In accord with present findings, earlier reports using retrograde (Peschanski and Besson, 1984; Holstege, 1987) or anterograde (Veazey et al., 1982; Ho- soya, 1985) tracers demonstrated moderately dense PH projections to RM.

The NGCa (terminology of Andrezik and Beitz, 1985) lies lateral to RMIRPA, ventral to NGC and dorsal to the pyramidal tracts. Several studies, including the present one, have shown that PHIPH area is the source of light-to- moderate projections to NGCa (Peschanski and Besson, 1984; Hosoya, 1985; Holstege, 1987; Luppi et al., 1988).

Two functions commonly associated with nuclei of the ventromedial medulla (RM, NGCa, and to some extent RPA) are antinociception and the muscle atonia of rapid eye movement (REM) sleep (Peschanski and Besson, 1984; Lai and Siegel, 1988; Luppi et al., 1988; Gebhart and Randich, 1990; Vertes, 1990; Holmes and Jones, 1994). PH projections to the ventromedial medulla could serve a modulatory role in these or other functions.

Ventrolateral medulla [parvocellular reticular formation; nucleus paragigantocellularis lateralis; rostral uen- trolateral medullary region (RVLM)]. We showed that fibers originating from the caudal PH distribute moderately densely throughout the ventrolateral medulla to the PCR, dorsally, and to the RVLM region, ventrally. In accord with present findings, an early report in the rat (Ter Horst et al., 1984) as well as a later one in the cat (Fort et al., 1994) demonstrated significant numbers of labeled cells in the PH area following retrograde tracer injections in PCR/RVLM region.

Shammah-Lagnado et al. (1992) described considerably fewer PH projections to PCR in the rat than reported previously by Ter Horst et al. (1984) and proposed that differences may involve relative placements of retrograde tracers in the PCR; i.e., their injections (Shammah- Lagnado et al., 1992) were largely situated dorsally in PCR whereas those of Ter Horst et al. (1984) were primarily located ventrally in PCR. This distinction is consistent with present results showing that PH fibers distribute more densely to ventral (including the RVLM region) than to dorsal parts of PCR (see Figs. 5,6).

We found that PH projects lightly to the nucleus paragigantocellularis lateralis (PGN), medial to RVLM. Although previous reports (Lovick, 1985a; Ciriello et al., 1986; Van Bockstaele et al., 1989) have described significant PGN projections from the hypothalamus, most appeared to


originate from the dorsomedial, lateral, and paraventricular nuclei of the hypothalamus and few from PH.

Recent evidence indicates that rostro-ventrolateral medulla serves as a final common pathway between the forebrainiupper brainstem and lower brainstem and spinal cord systems involved in the control of various functions including cardiovascular activity, respiration, REM atonia. orofacial movements, and antinociception (Martin et al., 1981; Ross et al., 1984; Lovick 1985b, 1986; Ciriello et al.. 1986; Barman and Gebber, 1987; Siddall and Dampney, 1989; Guyenet, 1990; Castillo et al., 1991a,b; Ter Horst et al., 1991; Zagon and Smith, 1993; Dobbins and Feldman, 1994; Fort et al., 1994; Siddall et al., 1994). PH projections to the ventrolateral medulla may modulate some or all of these functions.

With respect to central cardiovascular systems, it is well documented that cell groups of the lower brainstem, particularly those of the lateral medullary tegmentum, exert a direct influence on autonomic visceromotor sites controlling cardiovascular activity (Ciriello et al., 1986; Guyenet, 1990; Saper, 1995). This has recently been substantiated using the transneuronal transport of viruses. Injections of pseudorabies virus into the heart gave rise to labeled (infected) cells in vagal preganglionic motoneurons of the dorsal motor nucleus (of vagus) (DMV) and the nucleus ambiguus (NA) as well as cells in several brainstem sites with presumed input to the DMV and NA such as the nucleus of the solitary tract, the medullary lateral tegmental field (parvocellular RF and RVLM), the caudal raphe (magnus, pallidus, and obscurus), PGN, the dorsolateral pons (LC, LPB, and KF), and the pontomesencephalic PAG (Ter Horst et al., 1993; Standish et al., 1994, 1995). In a similar manner, injections of viruses into the stellate ganglion, the thoracic cord, or the adrenal gland produced transneuronal labeling of brainstem sympathetic premotor neurons including those of the rostral ventrolateral and ventromedial medulla, the lateral pons (A5 area) and the pontomesencephalic PAG (Strack et al., 1989; Jansen et al., 1995a,b). The present findings that the PH (mainly the caudal PH) projects to several vagalisympathetic premotor nuclei suggests that PH is critically involved in autonomic regulation.

We demonstrated that caudal PH fibers distribute densely to all three major subdivisions of the 10: principal, medial accessory, and dorsal accessory olive. Several studies in the rat (Brown et al., 1977; Carlton et al., 1982; Peschanski and Mantyh, 1983; Swenson and Castro, 1983a,b; Bentivoglio and Molinari, 19841, cat (Saint-Cyr and Courville, 1981; Walberg, 1981), and opossum (Henkel et al., 1975; Linauts and Martin, 19781, using anterograde and/or retrograde tracers, have identified projections to I 0 from a region of the caudal diencephalon below the fasciculus retroflexus, variously termed the subparafascicular nucleus (Swenson and Castro, 1983a; Bentivoglio and Molinari, 19841, the subparafascicular area (Peschanski and Mantyh, 19831, or the nucleus parafascicularis prerubralis (Carlton et al., 1982). The subparafascicular area (Peschanski and Mantyh, 1983) overlaps with PH, suggest- ing that some of the previously described subparafascicular projections to I 0 may have originated from PH.

In accord with our findings of caudal but not rostral PH projections to 10, Hosoya (1985) failed to observe rostral PH-area projections to the 10. Although the functional significance of PH (or caudal diencephalic) projections to

Inferior olive.

DESCENDING PROJECTIONS OF PH 62 7

the I0 is unknown, it has been proposed that they may play a role in complex motor coordination (Swenson and Castro, 1983b; Bentivoglio and Molinari, 1984).

Control injections Control injections were made in the VTA, SUM, dorsal

premammillary (PMd), dorsomedial (DMh), and lateral (LHy) nuclei of the hypothalamus. VTA, SUM, PMd, and DMh projections to the brainstem were considerably less dense and widespread than those from PH. For the most part, projections from each of these sites were confined to the upper brainstem.

VTA fibers predominantly terminated within ventral parts of the pontomesencephalic gray (ventral CG, LDT, and LC) and midline structures (DR, MR, raphe pontis, and nucleus incertus). This pattern of projections is in general agreement with previous reports showing that VTA projects to the PAG, DR, MR, LC, and parts of the dorsolateral pons (Swanson, 1982; Fallon and Loughlin, 1985). Consistent with previous findings (Vertes, 19921, the main targets of SUM fibers were the lateral/ventrolateral PAG and the dorsal raphe nucleus.

The pattern of brainstem projections from PMd and DMh was similar. Both the DMh and PMd were found to project strongly to the dorsolateral PAG, and significantly but less densely to the dorsolateral pons (PPT, LPB, and CUN), the dorsal and median raphe nuclei, and the LC. These findings are in essential accord with previous results (Ter Horst and Luiten, 1986; Luiten et al., 1987; Canteras and Swanson, 1992), with the exception that we observed considerably fewer DMh projections to the lower brainstem than shown by Ter Horst and Luiten (1986). Unlike that report, however, we did not systematically examine the DMh, and as such, our DMh injections may not have included that region of DMh that projects to the caudal brainstem.

We found that the projections from the lateral hypothalamus to the brainstem were considerably stronger than those from other sites bordering PH. In accord with previous examinations of LHy projections to the brainstem (Saper et al., 1979; Hosoya and Matsushita, 1981; Berk and Finkelstein, 1982; Holstege, 1987; Luiten et al., 1987; Villalobos and Ferssiwi, 1987; Allen and Cechetto, 19921, we showed that LHy fibers distribute moderately to heavily to several structures throughout the brainstem including the lateral PAG, superior colliculus, lateral pontomesencephalic RF, SNC, PP, DR, PPT, LC, lateral parabrachial nucleus, medial and lateral medullary areas, and the nucleus of the solitary tract. Although LHy and PH were found to project to some common sites, their patterns of brainstem projections were largely distinct; that is, PH projects much more heavily to medial than to lateral regions of the brainstem and the reverse is true for LHy.

General functional considerations The posterior hypothalamic nucleus has been linked to

several functions including cardiovascular activity, respiration, locomotion, wakefulnessiarousal, antinociception, and recently the modulation of the hippocampal EEG (Shik and Orlovsky, 1976; Buccafusco and Brezenoff, 1979; Eldridge et al., 1981, 1985; Carstens, 1982; Ohta et al., 1985; DiMicco et al., 1986; DiMicco and Abshire, 1987; Mori, 1987; Martin et al., 1988; Waldrop et al., 1988; Wible et al., 1988; Lin et al., 1989; Pare et al., 1989; Spencer et al., 1990; Sinnamon, 1993; Bland et al., 1993, 1995; Vertes et al.,

1995). The PH has been viewed as an important center for the integration of these various activities. Waldrop and colleagues showed in an early report (Eldridge et al., 1981) that stimulation within the PH area in unanesthetized decorticate cats produced parallel increases in locomotion, respiration, and blood pressure and, subsequently (El- dridge et al., 1985; Waldrop et al., 1988), that microinjections of the GABA antagonists, bicuculline or picrotoxin, into PH of anesthetized cats resulted in increases in blood pressure, heart rate, minute ventilation, and limb movements. They concluded that PH plays a role in regulating cardiorespiratory activity during exercise.

In a similar manner, DiMicco and co-workers have shown 1) that microinjections of GABA antagonists into PH of urethane-anesthetized or conscious rats produced increases in heart rate, blood pressure, and sympathetic nerve activity (DiMicco et al., 1986; DiMicco and Abshire, 1987; Wible et al., 1988); 2) that injections of GABA agonists into PH of conscious rats resulted in reductions in each of these responses (Wible et al., 1988); and 3) that injections of GABA antagonists into the PH area in conscious rats elicited a set of behaviors (intermittent running, rearing, and jumping) that were interpreted as components of escape or flight behavior (Shekhar and DiMicco, 1987; Wible et al., 1988). Others have also reported that electri- cally (Yardley and Hilton, 1986; Lammers et al., 1988) or chemically induced (with GABA antagonists) (DiScala et al., 1984; Schmitt et al., 1985) activation of PH/PH area produces a pattern of locomotion resembling flightiescape.

The presently described descending projections of PH are consistent with a role for PH in complex functions (e.g., exercise, defensive behaviors) involving several components including attentioniarousal, respiration, autonomic, and somatomotor activity. PH projections to the dorsolateral pons/pontomesencephalic RF could serve a role in hippocampal and cortical EEG activation; those to the medial pontomedullary RF in locomotor activity; those to the PAG in defensive and antinociceptive behaviors; and those to the lateral reticular tegmentum and rostral ventromediali ventrolateral medulla in REM atonia and cardiorespiratory functions.

We recently examined the ascending PH projections in the rat (Vertes et al., 1995) and showed that PH fibers distribute densely to several “limbic-related” subcortical and cortical sites and to structures with direct links to the hippocampus. Based on our analysis of ascending PH projections, we proposed that PH may be critically involved in various aspects of emotional/motivational behaviors including mnemonic processes associated with significant emotional events (Vertes et al., 1995). The present demonstration of descending PH projections to brainstem structures regulating visceral and somatomotor activity further supports an essential role for PH in the control of complex behaviors involving an integration of respiratory, visceromotor, and somatomotor activity.

ACKNOWLEDGMENTS We thank Sherri Von Hartman for excellent graphic

work. This research was supported by grants MH45075 and NS35883 to R.P. Vertes.


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Descending projections of the posterior nucleus of the hypothalamus:Phaseolus vulgaris...

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