THE JOURNAL OF COMPARATIVE NEUROLOGY 299:10&122 (1990)

Neuronal ConnectionsBetween the Cerebellar Nuclei and Hypothalamus in M;acuuxc Fascicuktris.. Cerebello-Visceral Circuits D.E. HAINES, P.J. MAY, AND E. DIETRICHS Department of Anatomy (D.E.H.)and Departments of Anatomy and Ophthalmology (P.J.M.), The University of Mississippi Medical Center, Jackson, Mississippi 39216, and Anatomical Institute, University of Oslo, Oslo, Norway (E.D.)

ABSTRACT The purpose of this study was to identify the basic pattern of interconnections between the cerebellar nuclei and hypothalamus in Macuca fusciculuris. The distribution of retrogradely labeled cells and anterogradely filled cerebellofugal axons in the hypothalamus of M . fuscicularis was investigated after pressure injections of a horseradish peroxidase mixture (HRP + WGA-HRP) in the cerebellar nuclei. Following injections in the lateral, anterior, and posterior interposed cerebellar nuclei retrogradely labeled cells were present in the following areas (greatest to least concentration): lateral and dorsal hypothalamic areas, dorsomedial nucleus, griseum periventriculare hypothalami, supramammillary and tuberomammillary nuclei, posterior hypothalamic area, ventromedial nucleus and periventricular hypothalamus, around the medial mammillary nucleus, lateral mammillary nucleus, and infundibular nucleus. Cell labeling was bilateral with an ipsilateral preponderance. In these same experiments anterogradely labeled cerebellar efferent fibers terminated in the contralateral posterior, dorsal and lateral hypothalamic areas, and the dorsomedial nucleus. In these regions retrogradely labeled hypothalamic cells were occasionally found in areas that also contained anterogradely filled cerebellar axons. This suggests a partial reciprocity in this system. In addition, sparse numbers of labeled cerebellar fibers recross in the hypothalamus to distribute to homologous areas ipsilateral to the injection site. Subsequent to an injection in the medial cerebellar nucleus (NM), cell labeling was present in more rostral hypothalamic levels including the lateral and dorsal hypothalamic areas, the dorsomedial nucleus, around or in fascicles of the column of the fornix, and in the periventricular hypothalamic area. Although no fastigiohypothalamic fibers were seen in this study, on the basis of information available from the literature it is likely that such a connection exists in primates. In summary, hypothalamic projections to NM originated mainly from rostral to midhypothalamic levels, whereas those projections to the lateral three cerebellar nuclei came from mid and more caudal levels. The existence of direct hypothalamic projections to cerebellar nuclei in M . fascicularis and of cerebellofugal projection to some hypothalamic centers indicates that circuitry is present through which the cerebellum may influence visceral functions. Furthermore, the fact that projections to NM versus the other cerebellar nuclei originate from somewhat different regions of the hypothalamus would suggest that the visceral functions modulated by each pathway is not the same. Key words: cerebellum, cerebellohypothalamic, hypothalamocerebellar,visceromotor pathways

Accepted June 13, 1990. Address reprint requests Dr. D.E. Haines, Department of Anatomy, The University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. B'

1990 WILEY-LISS, INC.

CEREBELLAR-HYPOTHALAMICINTERCONNECTIONS It is well known that the cerebellum receives somatically derived afferent information and, through its efferent connections, influences the activity of skeletal muscles (e.g., Bloedel, '73; Bloedel and Courville, '81; Brooks and Thach, '81; Ito, '84; Anderson et al., '87). In agreement with this, cerebellar dysfunction is usually characterized by varying degrees of muscle asynergy (Gilman et al., '81).In contrast, stimulation or ablation of cerebellar structures have produced a variety of visceral responses including piloerection, changes in blood pressure, heart rate and respiration, alterations in smooth muscle tone (bladder, pupil, intestines, nictitating membrane), urination, and increased cerebral blood flow (e.g., Moruzzi, '40; Wiggers, '43; Chambers, '47; Zanchetti and Zoccolini, '54; Ban et al., '56; Raymond, '58; Sawyer et al., '61; Ban, '64; Miura and Reis, '69, '70; Achari et al., '73; Dow, '74; DelBo et al., '83; Nakai et al., '83; Nisimaru and Kawaguchi, '84; Nisimaru and Watanabe, '85; DelBo and Rosina, '86; Chida et al., '86). These observations indicate a cerebellar role in visceral as well as skeletal motor function. The consensus has been that cerebellovisceral interactions are primarily mediated through multisynaptic circuits via the reticular formation (Ban, '64; Miura and Reis, '69, '70; Martner, '75; see Dietrichs and Haines, '89 for review). However, the demonstration of cerebellar projections from brainstem visceral centers (Kotchabhakdi and Walberg, '77; Somana and Walberg, '78, '79a,b; Saigal et al., '80; Zheng et al., '82; Shapiro and Miselis, '85) and of interconnections between hypothalamic and cerebellar structures (see Dietrichs and Haines, '89 for review) suggests that more direct cerebellovisceral circuits may exist. Studies with anterograde and retrograde tracing methods in rat, cat, tree shrew, and prosimians and New World primates (Galago, Saimiri) have revealed hypothalamic projections to the cerebellum and some cerebellar nuclear projections into hypothalamic areas (Dietrichs, '84; Dietrichs and Haines, '84, '85a,b, '86; Haines and Dietrichs, '84, '87; Haines et al., '84, '85, '86). Such projections may also be present in nonmammalian vertebrates (Bangma and ten Donkelaar, '82; Kunzle, '83; Smeets and Boord, '851.' Although the general nature of this connection has been established, no corroborating data are available from Old World anthropoid primates and the patterns of hypothalamic labeling following placement of retrograde tracers in the cerebellar nuclei have only been investigated in the cat (Dietrichs and Haines, '85b; Dietrichs et al., '85). This latter point is of interest because fastigial stimulation produces cardiovascular and some behavioral responses (e.g., Miura and Reis, '69, '70; Reis et al., '73; Achari et al., '73; Nisimaru and Kawaguchi, '84; Chida et al., '86; Oomura et al., '89), whereas such responses have not been reported after exploration of the other cerebellar nuclei (Miura and Reis, '69, '70; Achari et al., '73; Reis et al., '73). This suggests a differential hypothalamic connection between the medial versus the more lateral cerebellar nuclei. With these previous data in mind, the present study has undertaken to use anterograde and retrograde transport of 'Bangma and ten Donkelaar ('82) reported retrograde cell labeling in the turtle (preoptic area, DHAr, periventricular nucleus) and lizard (DHAr) hypothalamus subsequent to injections of HRP in the cerebellum. They concluded, however, that these patterns might be related to the spread of the HRP from the injection site in the cerebellum to the locus coeruleus. In light of the confirmatory data of Kunzle ('831,it is our opinion that Bangma and ten Donkelaar showed a true hypothalamocerebellar projection in the turtle and lizard.

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horseradish peroxidase and a wheat germ agglutininhorseradish peroxidase conjugate (HRPWGA-HRP) in Macaca fascicularis. The following points were of particular interest: (1)What is the distribution pattern of retrogradely labeled hypothalamic cells following injections in the cerebellar nuclei? (2) Is there evidence of cerebellofugal projections in hypothalamic areas, and are any of these reciprocal to the distribution of retrogradely labeled cells?

Four male, young adult Macaca fascicularis monkeys (3.5-4.5 kg) were used in this study. Animals were initially sedated by administration of ketamine HCl(10 mg/kg, im) to allow placement of an intravenous catheter. Surgical anesthesia was then obtained by injection of sodium pentobarbital, (35 mg/kg, iv) and maintained with smaller intravenous doses, so as to eliminate skeletal muscle reflexes. All surgical procedures were done under aseptic conditions and in accordance with NIH guidelines. Dexamethasone (1 mgkg, iv) was given to reduce postsurgical inflammation. Upon awaking animals were given Meperidine (1 mg/kg, im) as an analgesic. Survival periods ranged from 20-26 hours. The animals were placed in a stereotaxic head holder, and one occipital pole of the cerebral cortex was removed by subpial aspiration to reveal the cerebellum. After the cerebellar surface was visualized and superficial landmarks identified, the tip of the syringe was positioned in the cerebellar nuclei according to stereotaxic coordinates (Szabo and Cowan, '84). Pressure injections of 0.12-1.0 p1 of a mixture containing 20% HRP and 1.0% WGA-HRP were made in each of the cerebellar nuclei using a 1.0 p1 Hamilton syringe. The representative medial nucleus (NM) and posterior interposed nucleus (NIP) cases described below had injections of 0.14 p1 and 0.32 pl, respectively. Following the survival periods, the animals were rendered unconscious with ketamine HCL (10 mgkg, im), catheterized, and given an overdose of sodium pentobarbital (100 mg/kg, iv). The animals were artificially ventilated with 95% oxygen and 5% carbon dioxide, given 2.0 ml Heparin (iv) and 1.0 ml of sodium nitrite (1.0%, iv) and perfused

Abbreviations

cc DHAr DMNu F GPH IN LHAT LMNu MMNu NIA NIP NL NM OpTr PHAr

PvH SMNu STNu TMNu VmNu I11 or IIh

crus cerebri dorsal hypothalamic area dorsomedial nucleus fornix griseum periventriculare hypothalamic (caudal periventricular grey) infundibular nucleus lateral hypothalamic area lateral mammillary nucleus medial mammillary nucleus anterior interposed cerebellar nucleus posterior interposed cerebellar nucleus lateral cerebellar nucleus medial cerebellar nucleus optic tract posterior hypothalamic area periventricular hypothalamic area supramammillary nucleus subthalamic nucleus tuberomammillary nucleus ventromedial nucleus third ventricle

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108 transcardially with 1,000 ml of saline, followed by 3,000 ml of fixative containing 1.0% paraformaldehyde and 1.25% glutaraldehyde in pH 7.4, 0.1 M phosphate buffer. After perfusion the brain was blocked stereotaxically, removed, and postfixed for 1 hour in the above fixative. The cerebellum was cut from the brainstem and placed in 30% sucrose in pH 7.4,O.l M phosphate buffer at 4°C and the brainstem was placed in the buffer alone at 4°C. The brainstem was cut into serial 50 km coronal sections on a vibratome; the cerebellum was frozen and cut into 50-pm horizontal or coronal sections on a sliding microtome. Both sets of sections were treated with a modification of the tetramethylbenzidine (TMB) method of Mesulam ( ' 7 8 ) . Briefly, the sections received three 5-minute washes in distilled water, were preincubated for 20 minutes in a solution containing 0.01% TMB, 0.1% sodium nitroprusside, 4.0% ethanol in a pH of 3.3 acetate buffer, and reacted for 20 minutes in the same solution following the addition of hydrogen peroxide (0.3%at 3.0 ml/lOO ml). The sections were then rinsed in and mounted out of dilute pH 3.3 acetate buffer. They were then stained with neutral red, dehydrated, cleared, and coverslipped. Polarizing, brightfield, and darkfield microscopy were employed to observe and record the presence of labeled cells and axon terminals in the hypothalamus. Every other section was mounted and labeled cells counted in each. Labeled structures that were obviously fragments of cell bodies were not counted. The total estimated cell numbers described below were arrived at by doubling this figure in the assumption that the intervening unmounted sections contain similar populations of labeled cells.

RESULTS In the four animals used in this study, injections were centered in each of the cerebellar nuclei. Whereas the deposition of enzyme encompassed significant portions of each nucleus, it also involved parts of the immediately surrounding white matter (see Discussion) and, in the case of the interposed nuclei, small contiguous portions of the directly adjacent nuclei. Two basic patterns of retrograde hypothalamic cell labeling were seen. The first, subsequent to injections in the more lateral of the cerebellar nuclei (anterior and posterior interposed LNIA, NIP] and the lateral [ NLj cerebellar nuclei), consisted of cell labeling in mid and more caudal levels of the hypothalamus. The second, seen after an injection of the NM, was the tendency for labeled cells to be sequestered in more rostral areas.

Lateral injection (centeredin N I P ) The distribution of retrograde cell labeling in the hypothalamus was fundamentally similar following enzyme injections in NL, NIP, and NIA. In light of this observation and the fact that anterogradely labeled cerebellofugal axons distributed to similar hypothalamic targets, the NIP case is described as representative of these experiments. The injection site was centered in the NIP (Fig. 1) and minimally involved caudal and medial portions of the NL; some enzyme may have also entered the most caudal parts of NIA. Based on the pattern of anterograde label in the dorsal thalamus (Chan-Palay, '77), fibers exiting the caudal portions of the hilus were also involved. The injection site consisted of a central core of dense staining and a halo of moderately stained tissue (Fig. lA,B); collectively these comprise the maximum extent of the effective uptake area

Fig. 1. Tracings of the cerebellar nuclei of Mucnca fasczcular~sfrom rostral (A) to caudal (B) showing the location and extent of the inlection centered in the posterior interposed nucleus (NIP).Scale = 2.0 mm.

(Walberg et al., '80; Ahlsen, '81; Mesulam, '82). Although it is acknowledged that some limited hypothalamic cell labeling may be related to cerebellar cortical afferents from the hypothalamus that course through the periphery of the injection site (see Discussion), the vast majority is the result of enzyme deposition in the NIP. Subsequent to a placement of tracer in the NIP, retrogradely labeled cells were present in a variety of hypothalamic areas and nuclei (Fig. 2A-H). The few cells present at the level of the infundibulum (Fig. 2A) were located in the dorsal hypothalamic area, rostral portions of the lateral hypothalamic area, and insinuated among the fibers of the fornix. No labeled cells were seen in the Macaca hypothalamus rostral to this level following injections in the lateral three cerebellar nuclei. Beginning at about the level of the infundibular nucleus and proceeding caudally, there was a marked increase in the number of retrogradely filled hypothalamic somata. Well-labeled cells were present in the ventromedial and dorsomedial hypothalamic nuclei (Figs. 2B-D, 3B,C), diffusely distributed throughout the lateral hypothalamic area (Figs. 2B-D, 3E), and located adjacent to, or within, the fascicles of the fornix (Figs. 2C, 3D). This latter population is sometimes referred to as the perifornical nucleus (or region). The majority of labeled neurons in these areas had multipolar or stellate-shape somata with well-filled primary and sometimes secondary dendrites. Retrogradely filled cells in the periventricular area (Figs. 2B,D, 4A,B) had, for the large part, elongated fusiformshape cell bodies with labeled processes issuing from the poles of the soma. Such labeled cells were frequently found close to the ventricular surface (Fig. 4B). At mid to more

Fig. 2. Tracings of the hypothalamus of M . fasciculuris in coronal section from rostral (A) to caudal (H) showing the distribution of retrogradely labeled cells (large dots-one dot equals one cell) and anterogradely filled cerebellofugal axons (small dots). The individual tracings are of single sections spaced about equally distant from each other between the level of the infundibulum and the posterior hypothalamic areajust caudal to the maminillary body. Note the distribution of anterogradely labeled axons. Scale = 2.0 mm.

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Figure 2

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Fig. 3. Photomicrographs of hypothalamic labeling in M . fasczcularis under polarized light (A, C, D) and brightfield illumination following an injection centered in the NIP. Retrogradely labeled cells (at arrowheads)are shown, in relation to the injection site, in the posterior hypothalamic area (A--contralateral), the dorsomedial nucleus (B,

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C-ipsilateral), the lateral hypothalamic area (D, E+ontralateral), and in the perifornical region (El. Retrogradely labeled cells were found in areas that also contained anterogradely filled cerebellar fibers (A). Scale = 150 pm for A-D; 225 pm for E.

CEREBELLAR-HYPOTHALAMICINTERCONNECTIONS rostral hypothalamic levels (Fig. 2B-D), it appears that the dorsomedial nucleus is a prime source of hypothalamic input to the cerebellar nuclei. Cell counts reveal that the dorsomedial nucleus contains a significantly greater number of labeled cells than does the ventromedial nucleus (see Fig. 6). At a level corresponding to about the rostral end of the medial mammillary nucleus (Fig. 2D,E) there is a further increase in the number of labeled cells found in the lateral hypothalamic area and labeling is present in adjacent nuclei. Retrogradely filled somata were found in and adjacent to the supramammillary nucleus (Figs. 2F,G, 4A, 5A), throughout the ventral half of the lateral hypothalamic area and in, or close to, the medial mammillary, lateral mammillary, and tuberomammillary nuclei. Labeled cells were not present in the medial mammillary nucleus proper, but were found ventral and/or ventrolateral to this cell group (Figs. 4C,D, 5A) and occasionally on its lateral border (Fig. 2F). Although the lateral mammillary nucleus contained a few retrogradely filled somata (Figs. 2H, 4D), such labeling was usually seen in cells on the periphery of this nucleus or insinuated between it and the medial mammillary nucleus (Fig. 4D). Labeled cells were also found in the tuberomammillary nucleus and in the adjacent regions of the ventral portion of the lateral hypothalamic area (Fig. 2G,H). At caudal levels, filled cells were located in the posterior hypothalamic area, in caudal parts of the dorsal hypothalamic area, and in the caudal periventricular grey (Figs. 2G,H, 3A). Anterograde labeling. As a result of this NIP injection (see Fig. 11, anterogradely labeled cerebellofugal axons passed through the superior cerebellar peduncle and its decussation to enter the contralateral diencephalon. Many of these labeled axons coursed in a dorsolateral direction (Figs. 2E-H, 5A) into the thalamic nuclei. From these main cerebellothalamic fascicles, anterogradely labeled fibers coursed in a ventral and ventromedial direction to enter dorsal, posterior, and lateral hypothalamic areas (Figs. 2E-H, 3A, 5A,B). This projection was moderate to central and more lateral regions of the dorsal hypothalamic area at caudal levels (Fig. 2G,H) and sparse to this area at rostral levels (Fig. 2B-D). Labeled cerebellofugal axons also filtered ventrally into the posterior hypothalamic area at caudal levels (Figs. 2G,H, 3A), into dorsal portions of the lateral hypothalamic area (Fig. 2D-F), and at more rostral levels clusters of clearly labeled axons were present in what we interpret as the lateral part of the dorsomedial nucleus at its interface with the adjacent lateral hypothalamic area (Figs. 2B-D, 5B). In addition to these observations on the hypothalamic distribution of cerebellar efferent fibers, two specific points merit comment. First, as shown in Figure 3A (see also Fig. 2H), retrogradely labeled cells were occasionally found in hypothalamic regions that also contained anterogradely filled cerebellofugal axons. This proximity of labeled cells to fibers was noted in the dorsal and posterior hypothalamic areas and occasionally in the dorsomedial nucleus. Second, small fascicles, or single axons, exited from larger bundles of labeled fibers within the hypothalamus and coursed in a dorsomedial direction (Fig. 5A,B--large arrows), crossed the midline dorsal to the ventricular space, and distributed to homologous regions of the contralateral dorsal and lateral hypothalamic areas (Fig. 2F-H). Although sparse, this pattern of labeling was consistently seen from section to section.

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Laterality and cell numbers. Subsequent to injections in the lateral three cerebellar nuclei, hypothalamic cell labeling was bilateral with an ipsilateral preponderance (Fig. 6). Whereas this is the case for most areas, retrograde labeling in the dorsomedial nucleus is nearly bilaterally symmetrical. In the NIP experiment the dorsomedial nucleus and dorsal and lateral hypothalamic areas are the primary sources of hypothalamocerebellar projections. Of all the hypothalamic structures that project to the cerebellar nuclei, these three not only make the greatest contributions (Fig. 6) but also receive some direct cerebellofugal input (Fig. 2). The number of labeled hypothalamic cells varied from less than 10 in the infundibular nucleus to hundreds in the lateral hypothalamic area (Fig. 6). Taken collectively just under 1,100 hypothalamic cells were labeled in this experiment.

Medial injection (centeredin NM) In one experiment the injection site was centered in the NM (Fig. 7A,B). Although the site appeared to cross the midline, there was no uptake from the contralateral NM. Proof of this was seen in the overlying cerebellar cortex. Retrogradely labeled Purkinje cells formed a distinct rostrocaudally oriented strip adjacent to the midline (the A zone) on the side ipsilateral to the injection (Fig. 8A). No Purkinje cell labeling was seen in the more lateral cortex on the ipsilateral side or in any region of the contralateral cortex. Retrogradely filled hypothalamic cells (Fig. 8B) were found at levels located primarily between the optic chiasm and the most rostral aspect of the medial mammillary nucleus (Fig. 9A,B); labeled cells were rarely seen at, or caudal to, levels associated with the mammillary nuclei (see Fig. 9C). Labeled somata were located in more dorsal regions of the lateral hypothalamic area (45 cells; 31 ipsi./l4 contra) at rostral levels, occasionally in the rostral parts of the dorsal hypothalamic area (24; 17/71, and in the dorsomedial nucleus (33; 20113). Labeled cells were also present adjacent to, or within, the fascicles forming the column of the fornix (19, 1217) and in the immediate periventricular region (11; 714). One labeled cell each was found in the contralateral posterior hypothalamus and in the ventromedial nucleus. No cell labeling was seen in any of the mammillary nuclei or in the lateral hypothalamic area at caudal levels. Anterogradely labeled cerebellofugal axons could not be identified in any hypothalamic areainucleus in this case. This observation is open to two interpretations (see Discussion). First, although unlikely, it is possible that the NM does not project to the hypothalamus. Second, it is possible that, for unknown technical reasons, insufficient anterograde transport took place in this animal for cerebellohypothalamic terminals to be visualized. As in the NIP injection, hypothalamic cell labeling was bilateral with an ipsilateral preponderance in this NM case. There were, however, fewer cells labeled (1092 versus 134) and they were located at more rostral hypothalamic levels when compared to the NIP case.

DISCUSSION Five points concerning the organization of cerebellarhypothalamic interconnections emerge from this study. (1) Hypothalamic cells in Mucaca were retrogradely labeled following injections of HRP cocktail in the cerebellar nuclei and there is evidence of a general topographic pattern.

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Figure 4

CEREBELLAR-HYPOTHALAMICINTERCONNECTIONS Rostra1 to middle hypothalamic areas projected to the NM, whereas middle and more caudal areas projected to the NIP, NIA, and NL. (2) Although many hypothalamic areadnuclei contained retrogradely labeled cells, the majority were concentrated in dorsal and lateral hypothalamic areas ipsilaterally and in the dorsomedial nucleus bilaterally. (3) Labeled cerebellofugal axons from lateral and interposed cerebellar nuclei coursed into the contralateral hypothalamus and were specifically concentrated in posterior, lateral, and dorsal hypothalamic areas and in the dorsomedial nucleus. (4) Some of these projections may be partially reciprocal since retrogradely labeled cells were occasionally found in areas where anterogradely filled cerebellar efferent axons were also located. ( 5 ) There was evidence that some cerebellohypothalamic fibers may recross the midline and distribute to homologous areas ipsilateral to their cells of origin.

Hypothahnocerebellarpatterns To the best of our knowledge the first physiological evidence of a direct connection between the cerebellum and hypothalamus was offered by Whiteside and Snider ('53). These authors described short-latency-evoked potentials in the hypothalamus following cerebellar cortical stimulation and postulated that this may signify a direct cerebellar cortico-hypothalamic pathway. However, subsequent studies of cerebellar cortical efferents (see Haines et al., '82; Haines, '89 for reviews) failed to identify such a connection. Since tracing studies revealed many labeled cells in those hypothalamic regions (see Dietrichs and Haines, '89 for review) from which Whiteside and Snider ('53) recorded, it has been suggested that these authors were antidromically driving direct hypothalamocerebellar cortical axons (Haines et al., '84; Haines and Dietrichs, '84). Whiteside and Snider ('53),Ban and Inoue ('57), and Ban et al. ('56) also reported some longer latency responses. Collectively these observations have led to the consensus that cerebellar interaction with the hypothalamus was probably mediated via multisynaptic pathways (Ban, '64; see also Martner, '75).' Although the present study does not address the question of multisynaptic channels, it does offer convincing evidence that the hypothalamus of Macaca projects directly to the cerebellum. Following cerebellar nuclear injections, cell labeling was bilateral with an ipsilateral preponderance in most areas (see Fig. 6), concentrated in dorsal and lateral hypothalamic areas and in the dorsomedial nucleus, and to a certain extent topographically organized. Labeled cells have been reported in comparable hypothalamic structures 'Although Harper and Heath ('73) concluded (their pp. 285,291) that the fastigial nucleus projected to the hypothalamus, the only mention of degeneration within the hypothalamus in their study was to . . degenerating fibers , , . along the course of the medial forebrain bundle." Since no termination sites within the hypothalamus are described, we feel that this study did not offer conclusive evidence of a direct fastigio-hypothalamic connection. I'.

Fig. 4. Photomicrographs of retrogradely labeled cells (at arrowheads) in the hypothalamus of M. fusciculuris under brightfield illumination following an injection centered in the NIP. Retrogradely filled cells are shown, in relation to the injection site, in the supramammillary nucleus and periventricular area (A, B-ipsilateral), around the medial mammillary nucleus (C-contralateral, D-ipsilateral), and in, and around, the lateral mammillary and tuheromammillary nuclei (C-E). B is a photomontage. Scale = 225 km for A-D; 150 pm for E.

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following injections of tracers that involved cerebellar cortex and nuclei (Dietrichs and Haines, '84-Galago; Haines and Dietrichs, '84-Sazmiri; Haines et al., '85Tupaia). These studies did not, however, differentiate hypothalamic cell labeling related to the cortex uersus the nuclei. Dietrichs et al. ( ' 8 5 - c a t ) specifically investigated the patterns of hypothalamic cell labeling following placements of crystalline WGA-HRP in the cerebellar nuclei. They reported that the NM received input from the lateral, posterior, and dorsal hypothalamic areas, the NIP from the lateral hypothalamic area, but that no hypothalamic cells were labeled following placements of tracer in the NIA or NL. The present study confirms these prior results and offers further evidence on three points. First, in Macacu (present study) it appears that all cerebellar nuclei receive direct hypothalamic input. Dietrichs et al. ('85) noted that the lack of hypothalamic labeling in their NIA and NL cases may have been related to the small WGA-HRP placements. These authors also reported that anterogradely filled axons were present in all the ipsilateral cerebellar nuclei, and in the contralateral NM, following WGA-HRP injections in the cat hypothalamus. This indicates that a hypothalamocerebellar nuclear projection, as reported here for Macaca, also exists in the cat. Second, our results in Macaca (present study) suggest that hypothalamic input to NM originates from more rostra1 levels, whereas that to the NIA, NIP, and NL arises mainly from cells in middle and caudal levels. A rostrocaudal to mediolateral topography was not described for the cat (Dietrichs et al., '85). However, it has been reported that HRP-injections in lobule V (Saimiri) and V+VI (Galago) labeled hypothalamic cells located somewhat more rostrally, whereas injections in the posterior lobe hemisphere (Saimirz) or lobule VII (Galago) labeled cells at slightly more caudal levels (Dietrichs and Haines, '84; Haines and Dietrichs, '84; Haines et al., '84; see also Dietrichs and Zheng, '84-cat); an overlap of these rostrocaudal patterns was noted. Also, ter Horst and Luiten ('86-rat) described a projection from only caudal portions of the dorsomedial hypothalamic nucleus to the cortex of the posterior lobe (ansiform lobule). Third, our results on Macaca support the view that hypothalamocerebellar nuclear projections in primates may originate from a wider variety of hypothalamic centers than in the cat. In addition to cell labeling in dorsal, lateral, and posterior hypothalamic areas (Dietrichs et al., '85-cat), retrogradely filled cells in Macaca (present study) were also found in dorsomedial and ventromedial nuclei, the supramammillary, tuberomammillary, and lateral mammillary nuclei, adjacent to the fornix, in the periventricular grey, and in the region of the infundibulum (see Fig. 6). The distribution of retrogradely labeled hypothalamic cells reported subsequent to cortical injections that also involved parts of the cerebellar nuclei (Haines and Dietrichs, '84-Saimiri; Dietrichs and Haines, '84-Galago; Dietrichs, '84-cat; Dietrichs and Zheng, '84-cat; Haines et al., '85-Tupaia) is similar to that observed in the present study. This suggests that hypothalamic projections to the cerebellar nuclei may arise from basically the same areas that also supply the cortex or represent collaterals from parent axons passing to the cortex. It could be argued that the hypothalamic cell labeling in the present study was the result of involvement of afferent fibers passing around the nuclei on their way to the cortex. However, tissue damage at the injection sites was minimal and the sites were centered in the nuclei and did not involve

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Figure 5

CEREBELLAR-HYPOTHALAMIC INTERCONNECTIONS significant parts of the perinuclear white matter. Also, it has been reported that hypothalamocerebellar cortical fibers radiate through the white matter in a diffuse manner (Haines et al., '86). It is unlikely that large numbers of hypothalamic cells would be labeled pursuant to involvement of the modest number of hypothalamic afferent fibers found adjacent to the nuclei. Whereas it is acknowledged that some hypothalamic cell labeling may represent projections to the cerebellar cortex, we interpret the vast majority of labeled cells seen in these Macaca experiments as indicating somata with axons, or axon collaterals, that terminate in the cerebellar nuclei. The transmitter(s1 specific to hypothalamocerebellar fibers has not been conclusively identified. However, for three reasons, histamine is one likely candidate. First, histaminergic fibers have been identified in the cerebellar nuclei (Airaksinen and Panula, '88-guinea pig; Airaksinen et al., '89-Tupaia) and histamine-positive cells are present in hypothalamic centers known to project to the cerebellum, namely, the dorsomedial, ventromedial, and supramammillary nuclei and the periventricular area (Airaksinen and Panula, '88-guinea pig) and the tuberomammillary nucleus (Panula et al., '89-rat; Airaksinen et al., '89-Tupaia). Using fast blue and immunocytochemistry, Ericson et al. ('87-rat) reported L-histidine decarboxylasecontaining fast blue labeled cells in the tuberomammillary nucleus after injections in the cerebellar cortex plus parts of the interposed nuclei. Second, histamine-containing fibers are moderate (Panula et al., '89-rat) to numerous (Airksinen et al., '89-Tupaia) in midbrain periventricular areas; this appears to be the route followed by hypothalamic axons passing to the cerebellum (Haines et al., '86; ter Horst and Luiten, '86; Haines and Dietrichs, '87). Third, histaminergic fibers are found mainly in the cortex of the vermis and the flocculus and are present in molecular and granular layers (Airksinen and Panula, '88-guinea pig; Panula et al., '89-rat; Airksinen et al., '89-Tupaia; Panula et a1.-human, personal communication). Their distribution within the cerebellar cortex (as multilayered fibers, see Dietrichs and Haines, '%a, '89; Haines et al., '86; Haines and Dietrichs, '87) closely mirrors that seen in studies that have used anterograde tracing methods to identify hypothalamocerebellar fibers (Dietrichs and Haines, '85a; Haines et al., '86; ter Horst and Luiten, '86). The work of Panula and coworkers (e.g., Airaksinen and Panula, '88; Airaksinen et al., '89; Panula et al., '89a,b; Panula, unpublished data) suggest that the tuberomammillary complex may be the sole source of histaminergic fibers for the entire neuraxis. However, the existence of retrogradely labeled cells in nucleiiareas in addition to those of the tuberomammillary complex indicates that other, yet to be identified

Fig. 5. Low power photomontage (A) and photomicrograph (B) under polarized illumination showing anterogradely labeled cerebellar efferent fibers (A, B) and retrogradely filled cells (A-at white arrowheads) in the hypothalamus of M . fusczcularzs following an injection centered in NIP. From the larger bundles coursing into the dorsal thalamus labeled s o n s filtered ventrally into the dorsal and lateral hypothalamic areas (A+ontralateral) and a cluster of fibers are concentrated in the lateral aspects of the dorsomedial nucleus (Bcontralateral, note relationship to third ventricle). Filled axons exit both areas, course medially (A, B-large block arrows), cross the midline dorsal to the ventricular space, and distribute to homologous areas on the opposite side (see Fig. 2). Scale = 400 pm for A, B.

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transmitters may be present in this projection (see Dietrichs and Haines, '89 for review). The number of cells labeled in a given nucleusiarea is only a general indicator of the importance of the projection. However, the large number of labeled cells in the present study and their wide distribution are suggestive of a significant hypothalamocerebellar nuclear projection in Macaca. Although Dietrichs et al. ('85) did not specify cell counts, relatively few hypothalamic neurons were labeled in response to WGA-HRP implants in the cat cerebellar nuclei. In contrast, such data are available from reports on hypothalamic projections to the cerebellar cortex. In studies using HRP or WGA-HRP, a low of 18 labeled hypothalamic cells (paraflocculus injection) to a high of 222 cells (posterior lobe) have been reported (Dietrichs, '84; Dietrichs and Haines, '84; Haines and Dietrichs, '84). The observation of over 1,200 labeled cells (in the two cases described) in Mucaca is markedly higher than in previous reports that used comparable tracers and is open to three interpretations. First, transneuronal transport might account for the high cell counts. However, this is highly unlikely considering the short survival times, small injections, and method of tissue processing (see Wan et al., '82) used in the present study. Furthermore, Appenteng and Girdlestone ('87) noted that successful transneuronal labeling was only obtained after maximizing the sensitivity of the reaction product and that neurons so-labeled were invariably weakly labeled in comparison to other positive neurons. Second, it is possible that more hypothalamic cells project to the cerebellar nuclei than to the cortex, or that their terminals are simply more concentrated in a smaller area and therefore more available to the injected enzyme. Third, this pathway may be more developed in animals with larger, more complex brains. This view is given some credence by the observation of only 18labeled hypothalamic cells following injection of 0.3 pl of WGA-HRP in the paraflocculus of Galago (a prosimian primate) versus 59 labeled cells after placement of 0.3 ~1 of HRP in the same structure in Saimiri (a New World primate) (Dietrichs and Haines, '84; Haines and Dietrichs, '84). The observation of "a large number" of histamine-containing cells in the human tuberomammillary nucleus (Panula et al., '88) and of a denser plexus of histaminergic fibers in the human cerebellar cortex than in any other species studied to date (Panula, personal communication) further supports this concept.

Cerebellohypothalamicpatterns Whereas the general patterns of cerebellofugal projections are well known (e.g., Allen, '24; Voogd, '64; Angaut, '70; Angaut and Bowsher, '70; Earle and Matzke, '74; Martin et al., '74; Batton et al., '77; Chan-Palay, '77; Carpenter and Batton, '82; Asanuma et al., '83a,b), the presence of a direct cerebellar projection to hypothalamic structures has rarely been mentioned or illustrated. Wallenberg ('05) described degeneration in the hypothalamus using the Marchi method following a lesion that involved the superior cerebellar peduncle. Subsequent to lesions of the NL and interposed nuclei, anterograde degeneration was described in the "dorsolateral hypothalamus" (Cohen et al., ' 5 8 - c a t ) or in the "dorsolateral hypothalamic region" (Jacobs, '65-Tarsius, Marmoset). Cohen et al. ('58) specifically noted that the lateral and interposed nuclei did not project to "the main nuclear masses of the hypothalamus." Martin et al. ('74-opossum) described degeneration in the

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z

I-W

lpsilateral

@ Contralateral

DmNu PvH

DHAr SMNu LHAr TMNu LMNu MMNu PHAr

0

50

100

150

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Number of Cells Fig. 6. Bar graph showing the number of labeled cells in specific regions of the hypothalamus of M. fusciczduris ipsilateral and cantralateral to a n injection centered in the NIP. The cells indicated in relation to the medial mammillary nucleus (MMNu) were found on the immediate periphery of this nucleus and rarely within its borders proper. See text for discussion.

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Fig. 7. Tracings of the cerebellar nuclei of M. fusciculuris from dorsal (A) to ventral (B) showing the injection centered in the NM. Scale = 2.0 mm.

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Jansen, '72 and Chan-Palay, '77 for review^).^ Anterogradely labeled fibers from NM to hypothalamic centers were not seen in our only fastigial injection. These data are open to two interpretations. First, it is possible that the NM ofMacaca does not project to the hypothalamus. Carpenter and Batton ('82 for reviews), although noting that the rhesus NM projects to several nuclei of the dorsal thalamus, did not describe NM-to-hypothalamus fibers in this species. In contrast, it is possible that this projection is present in Macaca but, far technical reasons, was not labeled in our case. In this regard, an NM-to-hypothalamus connection has been reported in the opossum (Martin et al., '74anterograde degeneration), cat (Dietrichs and Haines, '85b-anterograde WGA-HRP), and squirrel monkey (Hainesand Dietrichs, '84-retrograde HRP). Also, Oomura et al. ('Bg-rat), Min et al. ('89-rat), and Katafuchi and Koizumi ('90-rat) have reported that the LHAr and paraventricular neurons receive a monosynaptic inhibitory input from the NM. In this Macaca study, retrogradely labeled hypothalamic cells were found in portions of the dorsal and posterior hypothalamic areas and dorsomedial nucleus that also contained filled cerebellofugal axons. Whereas these labeled axons and cells were not intimately apposed to each other, these data do suggest that some cerebellohypothalamic projections may be reciprocal with a segment of the hypothalamocerebellar component. Comparable partial overlap of labeled cells and fibers in the hypothalamus have also been reported in Galago and cat (Dietrichs and Haines, '84, '85b).

"lateral hypothalamic area" following destruction of the NM. Collectively these authors have indicated that this projection was relatively sparse and terminated contralatera1 to the lesion site. Functionalpoints The present study confirms these earlier anecdotal reports of cerebellar nuclear projections to hypothalamus, Experimental manipulation of the cerebellum, largely refines the general features of this pattern as described in through stimulation of the cortex and/or nuclei, has been other primates (Dietrichs and Haines, '84-Galago; Haines reported to result in a variety of visceral responses (see and Dietrichs, '84--Saimiri), the tree shrew (Haines et al., above; e.g., Moruzzi, '40, '50; Wiggers, '43a,b; Chambers, '851, and the cat (Dietrichs and Haines, '85b), and provides '47; Chambers and Sprague, '55a,b; Ban et al., '56; Raynew evidence of a direct cerebellohypothalamic projection mond, '58; Sawyer et al., '61; Achari et al., '73). Of in Macaca. Our Macaca data corroborate a lateral and particular interest is the fact that both vasopressor and interposed cerebellar nuclear projection to the contralateral vasodepressor responses have been reported following stimhypothalamus via the crossed ascending limb of the bra- ulation of the NM (e.g., Miura and Reis, '69, '70; Achari et chium conjunctivum (see also Cohen et d.,'58; Jacobs, '65). al., '73; Nisimaru and Kawaguchi, '84; Chida et al., '861, but These fibers are moderate to sparse and terminate in have not been seen following exploration of the other cerebellar nuclei (Miura and Reis, '69, '70; Achari et al., '73; dorsal, posterior, and lateral hypothalamic areas (Dietrichs Reis et al., '73). Miura and Reis ('70) suggested that the and Haines, '84; Haines and Dietrichs, '84; Dietrichs and vasopressor response is mediated through the reticular Haines, '85b; Haines et al., '85). The present report on formation and is functionally based on fastigial access to Macaca substantiates these prior observations and offers the sympathetic portions of the autonomic nervous system new evidence of a projection into the dorsomedial nucleus, a (see also Doba and Reis, '72; Nathan, '72; Nisimaru and connection not previously reported. In addition, it appears Kawaguchi, '84). Based on the autonomic responses seen that some anterogradely labeled fibers in Macaca recross subsequent to NM stimulation, on the anatomical observathe midline dorsal to the ventricular space and distribute to tions of Martin et al. ('74-opossum), Haines and Dietrichs dorsal and lateral hypothalamic areas ipsilateral to their ('84--SaZrniri), and Dietrichs and Haines ('85b-cat) and cells of origin. Although not described in studies on other on the physiological data of Oomura et al. ('Bg-rat), Min et primates (Dietrichs and Haines, '84; Haines and Dietrichs, al. ('89-rat), and Katafuchi and Koizumi ('go-rat), all '84) or the tree shrew (Haines et al., '851, a modest substantiating a direct NM-to-hypothalamus connection, it recrossing of these fibers has been reported for the cat is possible that this projection is a general feature of (Dietrichs and Haines, '85b). In contrast to Macaca, these mammalian vascular control. It has been shown that NM fibers in the cat appear to recross ventral to the caudal stimulation increases cerebral blood flow independent of portion of the ventricular space and distribute to only the increases in local cerebral metabolic levels (Nakai et al., dorsal hypothalamic area. This recrossing of some cerebello- '83), that such blood flow increases may be abolished by hypothalamic fibers in Macaca was not totally surprising in light of the fact that others have described and/or illus30nthe basis of the trajectoly of cerebellohypothalamicfibers, the possibiltrated a modest recrossing of cerebellothalamic axons (e.g., ity that some of these may be collateralsof cerebellothalamic axons must be Earle and Matzke, '74; Martin et al., '74; see Larsell and acknowledged (seealso Haines et al., '86).

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Fig. 8. Photomicrographs of retrogradely labeled Purkinje cells in the ipsilateral A zone (A-arrowheads) and in the hypothalamus (B-ipsilateral) under polarized illumination following an injection

centered in the NM. The large block arrow (A) indicates the approximate location of the midline. Note the obvious zonal configuration of Purkinje cell labeling (A). Scale = 400 pm for A; 150 pm for B.

lesions in the basal forebrain (Iadecola et al., '86a), and that this cerebral vasodilation may be mediated by cholinergic muscarinic receptors (Iadecola et al., '86b). The consensus is that NM initiated increases in cerebral blood flow are mediated through central pathways. The present results suggest that at least some of these vasoregulatory functions of the NM may be mediated through direct interconnections between the hypothalamus and fastigial nucleus (Haines and Dietrichs, '84; Dietrichs and Haines, '85b; Dietrichs et al., '85) as well as through the reticular formation (Ban, '64; Martner, '75). The more lateral of the cerebellar nuclei project to dorsal, posterior, and lateral hypothalamic areas and t o the dorsomedial nucleus and receive input back from these and other areas (present study; Haines and Dietrichs, '84;Dietrichs and Haines, '85; Haines et al., '86; see Dietrichs and Haines, '89 for review). In contrast to data on the NM, cardiovascular responses have not been reported following stimulation of the NIA, NIP or NL (Miura and Reis, '69, '70; Achari et al., '73; Reis et al., ' 7 3 ) . Whereas the interposed and NL may not have been methodically ex-

plored in this regard, it is reasonable to suggest that the projections of these more lateral cerebellar nuclei and their hypothalamic targets may be concerned with visceral functions that are not primarily vascular in nature. For example, the localization of glutaminase and aspartate aminotransferase in numerous cerebellar nuclear cells (Monaghan et al., '86-rat) indicates that many NL, NIA, and NIP-to-hypothalamus projections may be excitatory in contrast to the inhibitory action of fastigio-hypothalamic fibers as proposed by others (Min et al., '89-rat, Oomura et al., '89-rat, Katafuchi and Koizumi, '90-rat). Hypothalamic centers most obviously interconnected with the NIA,

Fig. 9. Tracings, in coronal section, of the hypothalamus of M . fusciculuris from rostral (A) to caudal ( C ) showing the distribution of retrogradely labeled cells (large dots-one dot equals one cell) following an injection centered in NM. Levels A and B are just rostral and caudal, respectively, to the infundibulum; level C is through the most caudal aspect of the medial mammillary nucleus. Scale = 2.0 mm.

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NIP, and NL (i.e., DHAr, LHAr, DMNu) contain cells that of the section shown in Figure 5B appeared in a recent project to a variety of forebrain centers (e.g., septa1 nuclei review. This work was supported, in part, by USPHS Grant and basal ganglia as broadly defined, amygdala, hippocam- EY07166 (to P.J.M.). pus) as well as to brainstem centers involved in a wide range of visceral functions (e.g., raphe nuclei, locus coeruleus, LITERATURECITED nucleus ambiguus, reticular formation, solitary complex, dorsal vagal nucleus, parabrachial nuclei) (e.g., Veazey et Achari, N.K., S. Al-Ubaidy, and C.B.B. Downman 11973) Cardiovascular responses elicited by fastigial and hypothalamic stimulation in conscious al., '82; Shen, '83; ter Horst and Luiten, '86). The dorsomecats. Brain Res. 60.439447. dial nucleus and the lateral hypothalamic area have been G. (1981) Retrograde labeling of retinogeniculate neurones in the cat implicated in the regulation of feeding behavior and in the Ahlsen, by HRP uptake from the diffuse injection zone. Brain Res. 223:374-380. modification of locomotion, sometimes accompanied by Airaksinen, M.S., and P. Panula (1988) The histaminergic system in the orofacial activity (e.g., Gladfelter and Brobeck, '62; Bellguinea pig central nervous system: An immunocytochemical mapping inger and Williams, '83; Bellinger et a]., '83; Moran et al., study using an antiserum against histamine. J . Comp. Neurol. 273163186. '83). Consequently, it is plausible to suggest that interconnections between the NL, N U , and NP and hypothalamus Airaksinen, M.S., G. Flugge, E. Fuchs, and P . Panula (1989) Histaminergic system in the tree shrew brain. J. Comp. Neurol. 286:289-310. may be involved in mediatingiregulating visceral activities, which are mutually coordinated with specific types of Allen, W.F. (1924) Distribution of the fibers originating from the different basal cerebellar nuclei. J. Comp. Neurol. 36:399-439. somatic responses. Anderson, G., D.M. Armstrong, S.A. Edgley, and M. Lidierth (1987) The cerebellar nuclei receive collaterals from a variety of Impulse activities of cerebellar neurones during locomotion in the cat. In afferent pathways, some are direct (e.g., spinocerebellar), J.S. King (ed): New Concepts in Cerebellar Neurobiology, New York: others are indirect (e.g., spino-olivo-cerebellar) (e.g., MatAlan R. Liss, pp. 349-370. sushita and Ikeda, '70; Hazlett et al., '71; Courville et al., Angaut, P. (1970) The ascending projections of the nucleus interpositus posterior of the cat cerebellum: An experimental anatomical study using '77; Robertson et al., '83; Dietrichs and Walberg, '87; Qvist, silver impregnation methods. Brain Rcs. 24:377-394. '89). Since the cerebellum projects to the hypothalamus, a relatively direct pathway exists through which somatically Angaut, P., and D. Bowsher (1970) Ascending projections of the medial cerebellar (fastigial) nucleus: An experimental study in the cat. Brain derived information can gain access to this visceral center. Res. 24.49-68. This could serve feedforward as well as feedback functions. Appenteng, K., and D. Girdlestone (1987)Transneuronal transport of wheat Feedforward in that somatically derived information could germ agglutinin-conjugated horseradish peroxidase into trigeminal interinform the hypothalamus, through afferent collaterals to neurones of the rat. J. Comp. Neurol. 258.387-396. the cerebellar nuclei and cerebellohypothalamic fibers, of Asanuma, C., W.T. Thach, and E.G. Jones 11983a) Distribution ofcerebellar terminations and their relation to other afferent terminations in the impending changes in the organism that may require ventral lateral thalamic region of the monkey. Brain Res. Rev. 5:2:37visceral responses. Feedback in that once a new functional 265. state is achieved it could be constantly monitored through C., W.T. Thach, and E.G. Jones (1983b) Anatomical evidence for cerebellohypothalamic and hypothalamocerebellar circuits. Asanuma, segregated focal groupings of efferent cells and their terminal ramificaNakai et al. ('83) have alluded to this possibility by suggesttions in the cerebellothalamic pathway of the monkey. Brain Res. Rev. ing that NM elicited increases in cerebral blood flow precede 5.267-296. increases in cerebral metabolic rates and that both prepare Ban, T. (1964) The hypothalamus, especially on its fiber connections and the septo-preoptico-hypothalamic system. Med. J Osaka Univ. 15:58-83 the organism for the demands placed on it by initiated vigorous activity. The same reasoning can be expanded to Ban, T., and K. Inoue (1957) Interrelation between anterior lobe of cerebellum and hypothalamus. Med. J. Osaka Univ. 7:841-857. include a wide range of autonomic responses. Indeed, the diffuse nature of hypothalamic projections to the cerebellar Ban, T., K. Inoue, S.Ozaki, and T.Kurotsu (1956) Interrelation between anterior lobe of cerebellum and hypothalamus in rabbit. Med. J. Osaka nuclei (and cortex) and the general pattern of cerebellar Univ. 7:lOl-115. projections to some hypothalamic centers would argue that Bangma, G.C., and H.J. ten Donkelaar (1982) Afferent connections of the cerebellar regulation of visceral functions is more global in cerebellum in various types of rcptiles. J. Comp Neurol. 207355-273. nature. Batton, R.R., A. Jayaraman, D. Ruggiero, and M.B. Carpenter (1977) Connections between the hypothalamus and cerebellar Fastigial efferent projections in the monkey: An autoradiographic study. J. Comp. Neurol. 174381-305. nuclei (and cortex) are not species-specificand represent an important channel through which a somatic regulating Bellinger, L.L., and F.E. Williams (1983) Aphagia and adipsia after kainic acid lesioning of the dorsomedial hypothalamic area. Am. J. Physiol. center can access the major visceral center in the brain244:R389-R399. stem. Concerning the potential of these observations, it is probable that the identification of connections between the Bellinger, L.L., L.L. Bernardis, and F.E. Williams (1983) Naloxone suppression of food and water intake and cholecystokinin reduction of feeding is cerebellum and visceral centers may ". . . lead us to a attentuated in weanling rats with dorsomedial hypothalamic lesions. conception of ataxia and asynergia in autonomic regulation Physiol. Behav. 31.839-846. similar to that now held for cerebellar disturbances in the Bloedel, J.R. (1973) Cerebellar afferent systems: A review. Progress in Neurobiol. 2.1-68. somatic sphere" (Fulton, '36).

ACKNOWLEDGMENTS The authors express their sincere appreciation to Ms. M. Danielson (technical assistance), Mr. T. Vickmark (some photography), Ms. D. Holmes (editorial assistance), and to Ms. G. Rainer and Ms. J. Brown (typing). We thank Dr. John D. Porter for his technical assistance and many helpful comments on the manuscript and Dr. Fred Walberg for his very relevant suggestions. A color photograph

Bloedel, J.R.. and J. Courvllle (1981) Cerebellar afferent systems. In V.B. Brooks (ed): Handbook ofPhysiology Section 1: T h e Nervous System Vol II, Bethesda: American Physiological Society, pp. 735-829. Brooks, V.B., and W.T. Thach (1981) Cerebellar control of posture and movement. In V.B. Brooks (ed): Handbook ofPhysio[ogy, Scction I : The Neruous System, Vol. I I . Bethesda: American Physiological Society, pp, 877-946.

Carpenter, M.B., and R.R. Batton, 111 (1982) Connections of the fastigial nucleus in the cat and monkey. In S.L.Palay and V. Chan-Palay (eds): The Cerebellum-New Vistas. New York: Springer-Verlag, pp. 251-295. Chambers, W.W. (1947) Electrical stimulation of the interior of the cerebellum in the cat. Am. J. Anat. 80:55-93.

CEREBELLAR-HYPOTHALAMICINTERCONNECTIONS Chambers, W.W., and J.M. Sprague (1955a) Functional localization in the cerebellum. I. Organization in longitudinal cortico-nuclear zones and their contribution to the control of posture, both extrapyramidal and pyramidal. J. Comp. Neurol. 103:105-129. Chambers, W.W., and J.M. Sprague (1955b) Functional localization in the cerebellum. 11. Somatotopic organization in cortex and nuclei. Archs Neurol. Psychiat., Chicago 74:653-680. Chan-Palay, V. (1977) Cerebellar Dentate Nucleus: Organization, Cytology and Transmitters. Berlin: Springer-Verlag, pp. 179-21 1,297-363. Chida, K., C. Iadecola, M.D. Underwood, and D.J. Reis (1986) A novel vasodepressor response elicited from the rat cerebellar fastigial nucleus: The fastigial depressor response. Brain Res. 370:378-382. Cohen, D., W.W. Chambers, and J.M. Sprague (1958) Experimental study of the efferent projections from the cerebellar nuclei to the brainstem of the cat. J. Comp. Neurol. 109:233-259. Courville, J., J.R. Augustine, and P . Martel (1977) Projections from the inferior olive to the cerebellar nuclei in the cat demonstrated by retrograde transport of horseradish peroxidase. Brain Res. 130:405419. DelBo, A., and A. Rosina (1986) Potential disynaptic pathways connecting the fastigial pressor area and the paraventricular nucleus of the hypothalamus in the rat. Neurosci. Lett. 71t37-42. DelBo, A., A.F. Sved, and D.J. Reis (1983) Fastigial stimulation releases vasopressin in amounts that elevate arterial pressure. Am. J. Physiol. 244:H687-H694. Dietrichs, E. (1984) Cerebellar autonomic function: direct hypothalamocerebellar pathway. Science 223:591-593. Dietrichs, E., andD.E. Haines (1984) Demonstration of hypothalamocerebellar and cerebello-hypothalamic fibres in a prosimian primate (Galago crassicaudatus).Anat. Embryol. 170:313-318. Dietrichs, E., and D.E. Haines (1985a) Do hypothalamo-cerebellar fibres terminate in all layers of the cerebellar cortex? Anat. Embryol. 173279284. Dietrichs, E., and D.E. Haines (1985b) Observations on the cerebellohypothalamic projection, with comments on non-somatic cerebellar circuits. Arch. Ital. de Biol. 123r133-139. Dietrichs, E., and D.E. Haines (1986) Do the same hypothalamic neurons project to both amygdala and cerebellum? Brain Res. 364241448. Dietrichs, E., and D.E. Haines (1989) Interconnections between hypothalamus and cerebellum. Anat. Embryol. 179t207-220. Dietrichs, E., and F. Walberg (1987) Cerebellar nuclear derents-where do they originate? A re-evaluation of the projections from some lower brain stem nuclei. Anat. Embryol. 177:165-172. Dietricbs, E., and 2-H. Zbeng (1984) Are hypothalamo-cerebellar fibers collaterals from the hypothalamo-spinal projection? Brain Res. 296225231. Dietrichs, E., F. Walberg, and D.E. Haines (1985) Cerebellar nuclear afferents from feline hypothalamus demonstrated by retrograde transport after implant.ation of crystalline wheat germ agglutinin-horseradish peroxidase complex. Neurosci. Lett. 54:129-133. Doba, N., and D.J. Reis (1972) Cerebellum: role in reflex cardiovascular adjustment to posture. Brain Res. 39:495-500. Dow, R.S. (1974) Some novel concepts of cerebellar physiology. Mount Sinai J. Med. 61:103-119. Earle, A.M., and H.A. Matzke (1974) Efferent fibers of the deep cerebellar nuclei in hedgehogs. J. Comp. Neurol. 154:117-132. Ericson, H., T. Watanabe, and C. Kohler (1987) Morphological analysis of the tuberomammillary nucleus in the rat brain: Delineation of subgroups with antibody against L-histidine decarboxylase as a marker. J. Comp. Neurol. 263:l-24. Fulton, J.F. (1936) The interrelation of cerebrum and cerebellum in the regulation of somatic and autonomic functions. In The Harvey Lectures, Vol. 31, Baltimore: Williams and Wilkins, pp. 135-182. Gilman, S., J.R. Bloedel, and R. Lechtenberg (1981) Disorders of the Cerebellum. Vol. 21 in Contemporary Neurology Series. Philadelphia: F.A. Davis. Gladfelter, W.E., and J.R. Brobeck (1962) Decreased spontaneous locomotor activity in the rat induced by hypothalamic lesions. Am. J. Physiol. 203.811-817. Haines, D.E. (1989) HRP study of cerebellar corticonuclear-nucleocortical topography of the dorsal culminate lobule-lobule V-in a prosimian primate (Galago):With comments on nucleocortical cell types. J. Comp. Neurol. 282274-292. Haines, D.E., and E. Dietrichs (1984) An HRP study of hypothalamocerebellar and cerebello-hypothalamic connections in squirrel monkey (Sairniri sczureus). J. Comp. Neurol. 229:559-575.

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Haines, D.E., and E. Dietrichs (1987) On the organization of interconnections between the cerebellum and hypothalamus. In: J.S. King (ed): New Concepts in Cerebellar Neurobiology. New York: Alan R. Liss, pp. 113-149. Haines, D.E., E. Dietrichs, and T.E. Sowa (1984) Hypothalamo-cerebellar and cerebello-hypothalamicpathways: A review and hypothesis concerning cerebellar circuits which may influence autonomic centers and affective behavior. Brain Behav. Evol. 24: 198-220. Haines, D.E., G.W. Patrick and P. Satrulee (1982) Organization of cerebellar corticonuclear fiber systems. In: S. Palay and V. Chan-Palay (eds): The Cerebellum-New Vistas. New York: Springer-Verlag, pp. 320-371. Haines, D.E., T.E. Sowa, and E. Dietrichs (1985) Connections between the cerebellum and hypothalamus in the tree shrew (Tupaiaglis).Brain Res. 328: 36 7-3 73. Haines, D.E., E. Dietrichs, J.L. Culberson, and T.E. Sowa (1986) The organization of hypothalamocerebellar cortical fibers in the squirrel monkey (Sazmiri sciureus). J. Comp. Neurol. 250:377-388. Harper, J.W., and R.G. Heath (1973) Anatomic connections of the fastigial nucleus to the rostra1 forebrain in the cat. Exp. Neurol. 39385-292. Hazlett, J.C., G.F. Martin, and R. Dom (1971) Spino-cerebellar fibers of the opossum Didelphis marsupials virginiana. Brain Res. 33:257-271. Iadecola, C., S. Mraovitch, M.P. Meeley, and D.J. Reis (1983) Lesions of the basal forebrain in rat selectively impair the cortical vasodilation elicited from cerebellar fastigial nucleus. Brain Res. 279:41-53. Iadecola, C., M.D. Underwood, and D.J. Reis (1986) Muscarinic cholinergic receptors mediate the cerebrovasodilation elicited by stimulation of the cerebellar fastigial nucleus in rat. Brain Res. 368:375-379. Ito, M. (1984) The Cerebellum and Neural Control. New York: Raven Press. Jacobs, V.L. (1965) The Cerebellofugal System in the Tarsius (Tarsiidae carbonarius) and the marmoset (Oedzpomidas oedApus). Doctoral dissertation, University of Kansas. Katafuchi, T., and K. Koizumi (1990) Fastigial inputs to paraventricular neurosecretory neurones studied by extra- and intracellular recordings in rats. J. Physiol. 421:535-551. Kotchabhakdi, N., and F. Walberg (1977) Cerebellar afferents from neurons in motor nuclei of cranial nerves demonstrated by retrograde axonal transport of horseradishperoxidase. Brain Res. 137:158-163. Kunzle, H. (1983) Supraspinal cell populations projecting to the cerebellar cortex in the turtle (Pseudemys scripta elegans). Exp. Brain Res. 49:l-12. Larsell, O., and J. Jansen (1972) The Comparative Anatomy and Histology of the Cerebellum, the Human Cerebellum, Cerebellar Connections, and Cerebellar Cortex. Minneapolis: Univ. of Minnesota Press, pp. 90-163. Martin, G.F., J.S. King, and R. Dom (1974) The projections of the deep cerebellar nuclei of the opossum, Didelphis rnarsupialzs uirginzana. J. Hirnforsch. 15545-573. Martner, J. (1975) Cerebellar influences on autonomic mechanisms. An experimental study in the cat with special reference to the fastigial nucleus. Acta Physiol. Scand. suppl. 425: 1-42. Matsushita, M., and M. Ikeda (1970) Spinal projections to the cerebellar nuclei in the cat. Exp. Brain Res. 10:501-511. Mesulam, M.-M. (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: A non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26:106-117. Mesulam, M.-M. (1982) Principles of horseradish peroxidase neurohistochemistry and their applications for tracing neural pathways-axonal transport, enzyme histochemistry and light microscopic analysis. In: Tracing Neural Connections with Horseradish Peroxidase. New York John Wiley & Sons, pp. 1-151. Min, B-I., Y. Oomura, and T. Katafuchi (1989) Responses of rat lateral hypothalamic neuronal activity to fastigial nucleus stimulation. J. Neurophysiol. 61: 1178-1 184. Miura, M., and D.J. Reis (1969) Cerebellum: a pressor response elicited from the fastigial nucleus and its efferent pathway in the brainstem. Brain Res. 13:595-599. Miura, M., and D.J. Reis (1970) A blood pressure response from fastigial nucleus and its relay pathway in brainstem. Am. J. Physiol. 219:13301336. Monaghan, P.L., A.J. Beitz, A.A. Larson, R.A. Altschuler, J.E. Madl, and M.A. Mullett (1986) Immunocytochemical localization of glutamate-, glutaminase- and aspartate aminotransferase-like immunoreactivity in the rat deep cerebellar nuclei. Brain Res. 363:364-370.

122 Moran, T.H., G.J. Schwartz, and E.M. Blass (1983) Organized behavioral responses to lateral hypothalamic electrical stimulation in infant rats. J. Neurosci. 3:10-19. Moruzzi, G. (1940) Paleocerebellar inhibition of vasomotor and respiratory carotid sinus reflexes. J. Neurophysiol. 3:20-31. Moruzzi, G. (1950) Problems in Cerebellar Physiology. Springfield, IL: Charles C. Thomas, pp. 3-116. Nakai, M., C. Iadecola, D.A. Ruggiero, L.W. Tucker, and D.J. Reis (1983) Electrical stimulation of cerebellar fastigial nucleus increases cerebral cortical blood flow without change in local metabolism: Evidence for an intrinsic system in brain for primaryvasodilation. Brain Res. 260:35-49. Nathan, M.A. (1972) Vasomotor projections of the nucleus fastigii to the medulla. Brain Res. 41:194-198. Nisimaru, N., and Y. Kawaguchi 11984) Excitatory effects on renal sympathetic nerve activity induced by stimulation at two distinctive sites in the fastigial nucleus ofrabbits. Brain Res. 304:372-376. Nisimaru, N., and Y. Watanabe (1985) A depressant area in the lateral nodulus-uvula of the cerebellum for renal sympathetic nerve activity and systemic blood pressure in the rabbit. Neurosci. Res. 3:177-181. Oomura, Y., B-I. Min, and T. Katafuchi (1989) Responses of rat lateral hypothalamic neuronal activity to fastigial nucleus stimulation. Neurosci. Abst. 15r1088. Panula, P., M.S. Airaksinen, U. Pirvola, and E. Kotilainen (1988) Histamineimmunoreactive neurons and nerve fibers in human brain. Neurosci. Abst. 14:211. Panula, P., U. Pirvola, S.Auvinen, and M.S. Airaksinen (1989b) Histamineimmunoreactive nerve fibers and terminals in the rat brain. Neuroscience 28:585-610. Panula, P., G. Flugge, U. Pirvola, S. Auvinen, and M.S. Airaksinen (1989a) Histamine-immunoreactive nerve fibers in the mammalian spinal cord. Brain Res. 484234-239. Qvist, H. (1989) Demonstration of axonal branching of fibers from certain precerebellar nuclei to the cerebellar cortex and nuclei: A retrograde fluorescent double-labeling study in the cat. Exp. Brain Res. 75:15-27. Raymond, A.M. (1958) Responses to electrical stimulation of the cerebellum of unanesthetized birds. J. Comp. Neurol. 110:299-320. Reis, D.J., N. Doba, and M.A. Nathan (1973) Predatory attack, grooming, and consummatory behaviors evoked by electrical stimulation of cat cerebellar nuclei. Science 182845-847. Robertson, B., G. Grant, and M. Bjorkeland (1983) Demonstration of spinocerebellar projections in cat using anterograde transport of WGAHRP, with some observations on spinomesencephalic and spinothalamic projections. Exp. Brain Res. 52:99-104. Saigal, R.P., A.N. Karamanlidis, J. Voogd, H. Michaloudi, and 0 . Mangana (1980) Cerebellar afferents from motor nuclei of cranial nerves, the nucleus of the solitary tract, and the nuclei coeruleus and parabrachialis in sheep, demonstrated with retrograde transport of horseradish peroxidase. Brain Res. 197200-206.

D.E. HAINES ET AL. Sawyer, C.H., J. Hilliard, and T. Ban (1961) Autonomic and EEG responses to cerebellar stimulation in rabbits. Am. J. Physiol. 200:405-412. Shapiro, R.E., and R.R. Miselis (1985) The central connections of the area postrema of the rat. J. Comp. Neurol. 234:344-364. Shen, C.L. (1983) Efferent projections from the lateral hypothalamus in the guinea pig: An autoradiographic study. Brain Res. Bull. 11:335-347. Smeets, W.J.A.J., and R.L. Boord (19851 Connections of the lobus inferior hypothalami of the clearnose skate Raja eglnnterra (chondrichthyes).J. Comp. Neurol. 234:380-392. Somana, R., and P. Walberg (1978) The cerebellar projection from locus coeruleus as studied with retrograde transport of horseradish peroxidase in the cat. Anat. Embryol. I55:87-94. Somana, R., and F. Walberg (1979a) Cerebellar afferents from the nucleus of the solitary tract. Neurosci. Lett. 11.4-47. Somana, R., and F. Walberg (197913) The cerebellar projection from the parabrachial nucleus in the cat. Brain Res. 172144-149. Szabo, J., and W.M. Cowan (1984) A stereotaxic atlas of the brain of the cynomolgus monkey (Macaca fascicularzs). J. Comp. Neurol. 222.265300. ter Horst, G.J., and P.G.M. Luiten (1986)The projections ofthe dorsomedial hypothalamic nucleus in the rat. Brain Res. Bull. 16:231-248. Veazey, R.B., D.G. Amaral, and W.M. Cowan (19821 The morphology and connections of the posterior hypothalamus in the cynomolgus monkey (Macacafascrcularis).11.Efferent connections. J. Comp. Neurol. 207:1S5156. Voogd, J. (1964) The CerebeIlum of the Cat, Structure and Fibre Connexions. Dissertation, University of Leiden, pp, 1-215. Walberg, F., T. Nordby, and E. Dietrichs (1980) A note on the anterograde transport of horseradish peroxidase within the olivocerebellar fibres. Exp. Brain Res. 40233-236. Wallenberg, A. (1905) Sekundare Bahmen aus dem frontalen sensibeln Trigeminuskerne des Kaninchens. Anat. Anz. 26:145-155. Wan, X-C.S., J. Trojanowski, and J.O. Gonatas (1982) Cholera toxin and wheat germ agglutinin conjugates as neuroanatomical probes: Their uptake and clearance, transganglionic and retrograde transport and sensitivity. Brain Res. 243.215-224. Whiteside, J.A., and R.S. Snider (1953) Relations of cerebeilum to upper brain stem. J. Neurophysiol. 16:397-413. Wiggers, K. (1943a) The influence of the cerebellum on the vegetative function. I. A review. Arch. neerl. De Physiologie27:286-300. Wiggers, K. (1943b) The influence of the cerebellum on the sugar content of the blood 111.Arch. neerl. De Physiologie 27:304-325. Zanchetti, A., and A. Zoccolini (1954) Autonomic hypothalamic outbursts elicited by cerebellar stimulation. J. Neurophysiol. 17t475-483. Zheng, Z-H., E. Dietrichs, and F. Walberg (19821 Cerebellar afferent fibers from the dorsal motor vagal nucleus in the cat. Neurosci. Lett. 32:113118.