The Organization of Feline Entopeduncular Nucleus Projections: Anatomical Studies KENNETH D. LARSEN

'' A N D RUSSELL L. MCBRIDE

Department of Anatomy, Emory Uniuersity, Atlanta, Georgia 30322

ABSTRACT The organization of entopeduncular nucleus (EPN) projections was studied in cats using autoradiographic and horseradish peroxidase (HRP) techniques. In autoradiographic studies, EPN axons were found to terminate in a J-shaped region in the dorsal and medial part of the ventral anterior nucleus (VA) and the rostral portion of the adjacent ventral lateral nucleus (VL). EPN axons also terminated in the rostral portion of the centrum medianum (CM), the ventrolateral portion of the lateral habenular nucleus (LHB), and the pedunculopontine nucleus (PP). The VA included the largest terminal field although the LHB had the greatest density of terminals. Regardless of the region of EPN into which amino acids were injected, the terminal fields were the same: there was no localization within the EPN of the cells projecting to one region. HRP-containing cells were distributed throughout the EPN following injections into the VA, LHB, or PP, although many more cells were labeled following injections into either VA or LHB than PP. EPN cells containing HRP following injections into either VA or LHB were not morphologically different from those not containing HRP in the same respective animals. Following HRP injections into stria medullaris, only cells in the rostral part of the EPN were labeled, providing evidence that rostrally and caudally located EPN neurons have different paths to LHB. Although there may be a rostrocaudal organization of pathways to LHB, individual regions of the nucleus project to the same areas. Clinical (Denny-Brown,'62) and experimental (Hore et al., '77) studies have demonstrated both postural and phasic motor deficits resulting from lesions of the basal ganglia. Although the mechanisms are not understood, the basal ganglia may influence movement through thalamocortical (Carpenter et al., '60; Frigyesi and Machek, '70) as well as noncortical paths (Newton and Price, '75). An understanding of the organization of the output of the basal ganglia is considered important in determining its function (Johnson and Clemente, '59; Nauta and Mehler, '66; Papez, '40; Ranson et al., '41; Wilson, '14; Woodburne et al., '46). The entopeduncular nucleus (EPN), the major output of the basal ganglia in carnivores, is the homolog of the primate medial pallidum: cell morphology (Fox et al., '661, inputs (Szabo, '62; Voneida, '601, and outputs (Carpenter and Strominger, '67; Kim et al., '76; Kuo and Carpenter, '73; Nauta, '74; Nauta and Mehler, '66) are the J.

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(19791 184: 293-308.

same. Studies of the output of the primate medial pallidum suggest a topographical organization of the pallidothalamic projections, with cells in the rostrodorsal and caudoventral portions projecting by way of the ansa and lenticular fasciculi, respectively, to different regions in the ventral tier thalamic nuclei (Kim et al., '76; Kuo and Carpenter, '73). Anatomical studies have not been performed, however, to localize cells within the EPN which project to different parts of thalamus or to non-thalamic regions (epithalamus and pons). Since cats are used widely for studies of motor systems, an understanding of the output organization of the feline basal ganglia is desirable. EPN projections were studied with autoradiographic and horseradish peroxidase techniques. The results show that each population ' Aided by NIH Grants N B 05669 and N S 06948. ' Present address: The Rockefeller University, New York. New York 10021. Portions of this work are part of a dissertation submitted in partial fulfillment of requirements for the Ph.D. degiee in physiology.

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of EPN cells projecting to different sites is distributed throughout t h e nucleus. Electrophysiological studies were also done to determine the projections of single EPN cells and their response to subthalamic nucleus stimulation, and were reported elsewhere (Larsen and Sutin, ' 7 8 ) .

2. Horseradish peroxidase experiments

A 50% solution of HRP (Type VI, Sigma) in saline was injected either unilaterally (3 cats) or bilaterally (5 cats) into t h e lateral habenular nucleus (LHB), unilaterally (3cats) or bilaterally (4 cats) into the region of t h e pedunculopontine nucleus ( P P ) , bilaterally into the stria terminalis (1 cat), bilaterally into t h e MATERIALS AND METHODS stria medullaris (1 cat), or unilaterally (2 Adult male and female cats weighing becats) or bilaterally ( 3 cats) into the VA or VL. tween 2.5 and 4.0 kg were used in all experi- The injection method was similar to t h a t used ments. The cats were anesthetized with 35 in t h e autoradiographic experiments. Injecmg/kg sodium pentobarbital for amino acid or tion volumes for t h e thalamic and LHB injechorseradish peroxidase (HRP) injections. tions were 0.1-0.2 pl and for t h e P P injections were 0.1-0.5 p1. 1. Au toradiographic experiments After survival times of two or three days, In autoradiographic experiments, 0.03-0.05 t h e cats were deeply anesthetized with sodium pl (100 pCIp1) of equal parts L-proIine-5-'H pentobarbital a n d perfused intracardially and D,L-(4,5-"H)-leucine (AmershamBearle) with saline followed by a buffered solution of were injected into different parts of t h e E P N 1.25%glutaraldehyde and 0.4% paraformaldeeither bilaterally (five cats) or unilaterally (1 hyde. The brains were blocked, removed from cat). A small hole was made in t h e cranium for t h e skull and stored overnight in fixative, and a direct vertical approach to t h e EPN. The then transferred to a buffered 30% sucrose injections were made using a 1-p1 Hamilton solution for 24 hours. The brains were then syringe (tip diameter of 50-100 pm) attached frozen and 40-pm sections were cut and prowith polyethylene tubing to a glass microcessed with diaminobenzidine (Jones and pipette. The exact volume to be injected was Leavitt, '74; Sutin and McBride, '77). Sections drawn into t h e pipette followed by 0.01 p l of from two of t h e brains with PP injections and oil to protect against leakage during insertion four with thalamic injections were also prointo t h e brain. The amino acid was injected in cessed with tetramethylbenzidine (Hardy and small increments over a 10-minute period. Heimer, '77; Heimer, personal communicaAfter three days of survival t h e animals tion). Cells were labeled more intensely with were deeply anesthetized a n d perfused with this method than with diaminobenzidine, but saline followed by lO!X formalin in saline. The since t h e numbers of labeled cells were simibrains were blocked, stored for three days in lar, the results are not reported separately. 30% sucrose-10% formalin, and cut in 25-pm Locations of labeled cells were plotted on sections on a freezing microtome. Each tenth projection drawings of cresyl violet-stained or twentieth section was mounted on albumin sections. coated microscope slides. The slides were then The proportion of HRP-containing E P N processed according to standard autoradineurons was estimated by dividing t h e numographic methods (Cowan e t al., '72) using ber of labeled neurons on five sections by the Kodak NTB-3 emulsion. After five weeks extotal number of E P N neurons on t h e same secposure, t h e slides were developed with Kodak tions. The presence of nucleoli could not be D-19 and stained with cresyl violet. The disused to prevent double-counting of neurons tribution of silver grains was studied with (Konigsmark e t al., '69) because t h e tissue bright- and dark-field microscopy and plotted was lightly stained with cresyl violet so t h e on projection drawings of t h e sections. The density of pathways or terminals was ex- faint nucleolus was obscured by t h e granular pressed as t h e number of silver grains in four reaction product. All HRP-containing cells adjacent 26 pm squares. The nuclear bound- were counted if they appeared to be reasonaries of Jasper and Ajmone-Marsan ('54) were ably intact, but t h e estimates of proportions adopted. Numerous investigators have illus- are not precise. trated t h e characteristic row or cluster of RESULTS silver grains found over axon fascicles (Con1. Autoradiographic experiments rad et al., '74; Cowan et al., '72; Edwards, '75; The autoradiographic experiments were deSwanson et al., '74).

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KENNETH D. LARSEN AND RUSSELL L. MCBRIDE

signed to compare projections from different ranged from 7 to 15 grains going from ventral parts of the EPN. Amino acid injections into to dorsal, respectively. the EPN were centered in the rostral (4 cases), The mammillothalamic tract is surrounded medial (2 cases), caudomedial (1case), caudal in part of its course by the VMT, which may be (2 cases), caudolateral (1 case), or lateral (1 differentiated from VL by its smaller, lighter case) part of the nucleus. Five injections were staining neurons (Rinvik, '68). Fibers from confined to the EPN; the other six encroached the EPN which traveled with this tract were slightly on either the preoptic region, medial confined near it a t caudal levels of VMT, but amygdaloid nucleus, reticular nucleus, or the separated from it a t more rostral levels and subthalamic nucleus. Regardless of the loca- distributed to VA. Therefore, the caudal VMT tion of the injection into the EPN, the fiber did not appear to have terminals; the rostral trajectories and terminal fields were similar. portion of VMT appears likely to have had terTherefore, one case will be described in detail. minals because silver grains were distributed The center of the injection site in cat L-62 is over it in continuity with those over VA. The fibers projecting to the centrum meshown in figure 1; this injection labeled the medial part of the EPN. The course of axons dianum (CM) approached the nucleus venand distribution of terminals from cat L-62 trally, and terminated primarily in the rostral are schematically represented in figure 2. portion of the CM. The region of greatest denAxons projecting to the ventral anterior nu- sity had 64 silver grains over the 2,700 pm' cleus (VA) took multiple paths. They coursed area. Silver grains over more caudal portions both through and around t h e ventral portion of the CM were interpreted t o be mainly of the internal capsule, and curved rostrally. over fibers passing through and destined for They then either ascended in the ventral me- the rostral CM or lateral habenular nucleus dial nucleus of the thalamus (VMT) to the me- (LHB). Because the boundaries of CM are indial portion of VA or continued in the rostral consistently defined (Jones and Leavitt, '74; pole of the reticular nucleus of the thalamus Mehler, '66), figure 4 shows photomicrographs where they arced back into the dorsal portion of the location of terminals from the lateral of VA. Axons also coursed through and around injection of L-65. the internal capsule and descended medial to The LHB terminal field had the greatest i t ; these axons arced around the zona incerta density of silver grains of all regions (146 per and then continued rostrally. They joined the 2,700 pm2).Axons projecting to the LHB took mammillothalamic tract and coursed ros- a wide path, most of which passed through the trally in and around it, then distributed to the medial VL to the internal medullated lamina, medial portions of VA and adjacent (rostral then through the mediodorsal nucleus of the and medial) ventral lateral nucleus (VL). The thalamus to enter and terminate in the LHB border between VA and VL is indistinct (Rin- ventrolaterally. Other fibers coursed adjacent vik and Grofova, '741, but silver grains were and lateral to the habenulo-interpeduncular mostly restricted to a J-shaped region, with tract, and some descended in the substantia the long arm of the J in the dorsomedial VA nigra and reticular formation to midbrain, (fig. 3) and the short arm in the ventromedial turned around, and coursed rostrally. These VA. The center of the terminal field had 120 latter fibers appeared to be the caudal portion silver grains in a 2,700 pm2 area (background of the fasciculus lenticularis, the ascending level of 3-7 for the same area), whereas VL limb of which joins other fibers in the H field. Abbreviations

AC, Anterior commissure AM, Anterior medial nucleus BC, Brachium conjunctivum C, Caudate nucleus CL, Central lateral nucleus CM, Centrum medianum CP, Cerebral peduncle EPN, Entopeduncular nucleus F, Fornix GP, Globus pallidus H, Fields of Fore1 IC, Internal capsule LG, Lateral geniculate nucleus

LHB, Lateral habenular nucleus MHB, Medial habenular nucleus MT, Mammillothalamic tract OT, Optic tract P, Putamen p, Paraventricular nucleus PO, Preoptic region PP, Pedunculopontine nucleus R, Reticular nucleus of the thalamus RN, Red nucleus SC, Suprachiasmatic nucleus SM, Stria medullaris SO, Supraoptic nucleus

ST, Stria terminalis STN, Subthalamic nucleus VA, Ventral anterior nucleus of the thalamus VB, Ventrobasal nucleus of the thalamus VL, Ventral lateral nucleus of the thalamus VM, Ventromedial nucleus of the hypothalamus VMT, Ventromedial nucleus of the thalamus 21, Zona incerta

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Fig. 2 Projections from t he EPN of cat L-62. Terminals are represented by small dots, axon8 cut in cross-section by large dots, and axons cut longitudinally by paired dots. The cross-hatching represents the injection site in figure 1. Although this brain was cut slightly obliquely in the frontal plane, the approximate level of each section according to the atlas of Jasper and Ajmone-Marsan (‘54) is indicated in the upper right of each.

Fig. 3 The location of terminals in VA. The enclosed area (a) includes part of VA, t h e internal medullated lamina and the anterodorsal nucleus, and is enlarged in darkfield illumination (b), taken at 30 X . The photomicrographs in c-e, taken a t 300 X , show the densities of silver grains in the ipsilateral doraomedial VA (c). the ipsilateral ventrolateral VA (d), and the contralateral dorsomedial VA (e).

Fig. 4 The location terminals in CM. The enclosed area (a) over the CM is enlarged in darkfield (b), taken a t 30 x . The typical morphology of the cells is shown (c), taken a t 300 x . Axons and terminals are schematically represented, as in figure 2, in d and e.

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Figure 5 shows the location of the terminals in the LHB ipsilateral (fig. 5c) and contralateral (fig. 5d) to the unilateral injection of L-62. There was no evidence of a path through stria medullaris or stria terminalis to LHB except for cases in which there was spread of amino acid to preoptic areas and the medial amygdaloid nucleus. In these cases, the fibers traveled in stria medullaris and entered and terminated dorsolaterally in the LHB, as well as ventrolaterally. Although s t r i a terminalis was labeled when injections spread to the amygdala, fibers could not be traced to LHB. Axons descending to the midbrain were most dense superior to and in the substantia nigra, pars reticulata, and lateral to the red nucleus, although they were difficult to follow because silver grains became sparse. Descending axons maintained a ventral position in the midbrain and moved dorsally to terminate mostly superior to but also inferior to the brachium conjunctivum at levels PO to P 1 in PP (17 silver grains/2,700 Fm'). Some fibers decussated in the supramammillary commissure of cat L-62 to ascend adjacent to the mammillothalamic tract. If contralateral thalamic terminals were present, they were too sparse to be recognized above background levels of silver grains. Sparse terminals were recognizable in the contralateral LHB (fig. 5d), the only observed contralateral terminal field, although the pathway could not be followed. Different parts of the EPN, which is three mm long by three mm wide, can be labeled by injections of the size used here. Figure 1, for example, shows an injection which intensely labeled the medial EPN a t the level of the caudal VM, and spared the lateral portion. In all 11 cases, the dorsomedial portion of VA is covered with silver grains, the LHB was typically densely covered in its ventrolateral portion, and silver grains were sparse over the PP, although fewer silver grains were over CM following the caudomedial injection than other rostral or lateral injections. Therefore, cells which project to each of the thalamic and non-thalamic sites are distributed throughout the nucleus. 2. Horseradish peroxidase experiments Bilateral (3 cats) or unilateral (2 cats) injections were made into VA and VL. Two injections were centered in VA, four in VL, one in the dorsal portions of VA and VL, and one in the medial part of VL and lateral VMT.

Regardless of the location of the injection, HRP cells were distributed throughout the rostrocaudal extent of EPN. Numerous cells were located among the fibers of the internal capsule dorsal and medial to the compact part of EPN (i.e., the EPN boundaries of Jasper and Ajmone-Marsan, '54). An injection (cat 372) which provided typical results is illustrated in figure 6A, and the distribution of labeled cells is plotted in figure 7. In this cat, 44% of the EPN cells were labeled. Labeled neurons were usually spindle shaped with several long dendrites. Regardless of the location of injection into LHB (dorsal, ventral or lateral portion) labeled cells were located throughout the EPN. Following unilateral injections almost all labeled EPN and lateral hypothalamic cells were ipsilateral to the injection; labeled cells contralateral to the injection averaged less than one per section. The distribution of labeled neurons after a unilateral injection in cat 218 into the lateral portion of LHB (fig. 6B) was typical, so the locations of HRP-containing somata in this animal a t levels rostral to LHB are plotted in figure 7. The neurons were distributed throughout the rostrocaudal extent of EPN, although fewer lateral EPN cells contained HRP. The greatest concentration of labeled neurons was located in the medial half of EPN and adjacent lateral hypothalamus, lateral and dorsolateral to the fornix. Many labeled neurons were also found among the fibers of the internal capsule lateral to the lateral hypothalamus, and dorsal to the EPN border of Jasper and Ajmone-Marsan ('54). The most rostral labeled cells were located in the medial and lateral preoptic region (fig. 7) and in or near the nucleus of the diagonal band. A few labeled cells were also found in VM. An average of 27 labeled neurons/section, representing 16% of the total population, was counted in the EPN, whereas the lateral hypothalamus typically contained 160 cells/section. Neurons labeled in the EPN and among the fibers of the internal capsule after LHB injections were often spindle shaped with several long dendrites. Cells located in the dorsal lateral hypothalamus immediately medial to the internal capsule, however, were usually smaller in size and more spherical in shape. HRP injections were made into stria terminalis and stria medullaris to determine if EPN cells projected by these routes to reach LHB. Following stria terminalis injections no EPN

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Fig. 7 Location of labeled neurons following unilateral injections into VA, LHB, and PP. Injection sites are in figure 6.

cells were labeled, and a n average of fewer than one lateral hypothalamic cell was labeled per section. Proof that these fibers took up the HRP was demonstrated by the observation of numerous labeled cells in the cortical and medial amygdaloid nuclei. These injections are discussed in greater detail in another publication (McBride and Sutin, '77). After bilateral stria medullaris injections, the distributions of labeled cells were similar on both sides of the brain. Marked cells were

most concentrated in the septum and were also in the preoptic region, the anterior lateral hypothalamus, and the rostral EPN. The average number of labeled cells was eighthection in the rostral portion of EPN, much less than that from LHB injections. The size of EPN cells which contained HRP following VA or LHB injections was estimated by multiplying the length of the soma by its width. When those EPN cells which were labeled by injections into either VA or LHB

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were compared with those which were not labeled in the same respective animals, the mean sizes were not different, and one-way analysis of variance showed that the distribution of sizes of labeled cells was not different from unlabeled cells. Following injection of HRP into the P P of cat 292 (fig. 61, labeled cells were distributed throughout the EPN, but most cells were in the rostral half of the nucleus (fig. 7). There was an average of three labeled cells per section in EPN, less than 1%of the cells. Labeled neurons were found also in the lateral hypothalamus medial to EPN, in the ventromedial hypothalamus including VM, and in the caudal hypothalamic area dorsal to t h e fornix. Bilateral injections into cat 257 included one (left side) which was similar to cat 292, and in which labeled cells were distributed throughout EPN, and one (right side) in which the injection was ventral to that on the left side and cells were located in the most medial part of EPN and in the ventromedial hypothalamus. Labeled EPN cells were large and generally spindle shaped, similar in morphology to EPN cells labeled after VA or LHB injections. The distribution of labeled EPN cells can be compared following VA, LHB, and PP injections. Although the labeled cells differed in distribution because of uptake by hypothalamic and other non-pallidal neurons and in number, in each case labeled EPN cells were distributed throughout the nucleus. No region of EPN included cells labeled from one nucleus but not another.

Nauta and Mehler, '66). However, the distribution of terminals in thalamus and the topographic organization is equivocal. Uno and Yoshida ('75) reported that EPN stimulation produced monosynaptic IPSPs in thalamic neurons located rostrally in VA or ventromedially in rostral VL regions, similar to the region in which the silver grains were most dense following the tritiated amino acid injections of this experiment. Using electron microscopy, Grofova and Rinvik ('74) reported degenerating terminals in VL but not in VA following EPN lesions. However, they did not specify the sites examined, and it is clear (fig. 2) that the EPN projection to VA and VL is not homogeneously distributed. Except for this electron microscopic study, anatomical and electrophysiological data are in agreement that there is an EPN input to VA. Our autoradiographic observations suggest that the EPN projection to the thalamus is primarily to VA. Kim et al. ('761, Kuo and Carpenter ('73), and Nauta and Mehler ('66) all report that the medial pallidum of primates projects t o VA and the oral and lateral, magnocellular parts of VL. Furthermore, primate pallidothalamic projections a r e topographically organized, both in fiber trajectories and termination. Both HRP and autoradiographic studies show that EPN projections to the thalamus arise from all parts of the nucleus, and that there is no point to point or topographical organization of the projection. Feline pallido-thalamic terminals appear to be more restricted than those of primates which involve more of VL. The projections of the thalamic region in DISCUSSION cats which receives the major EPN output Small tritiated amino acid injections were Le., the medial and dorsomedial regions of VA made to determine if different parts of EPN and VL) have been studied in degeneration exproject preferentially to different sites. Even periments. Kaelber ('70) reports that VA prothough most of the radioactive amino acid in- jects to CM, VL, parafascicular, and central corporated into protein is transported in the lateral nuclei of the thalamus, but does not reslow phase (Lasek, '681, 3-day survivals were port a cortical projection. Strick ('73) was chosen so that the silver grain pattern would unable to demonstrate a cortical projection reflect the terminal distribution more than from dorsal VA independent of fibers passing axonal pathways (Cowan et al., '72; Graybiel, through the nucleus, nor were movements '75; Swanson et al., '74). Nonetheless, axonal elicited by stimulation of this region. Small pathways were evident, as noted by other in- lesions in the ventrolateral half of VL provestigators (Conrad and Pfaff, '76; Cowan et duced terminal degeneration in area 4 of cats' al., '72; Graybiel, '75; Swanson et al., '74). motor cortex, and lesions of the dorsomedial half produced degeneration in area 6 of cortex Projections to thalamic nuclei (Strick, '73). Although Strick ('73) reported a The pallidal projection to ventral tier tha- somatotopic representation of the thalamolamic nuclei is well established (Carter and cortical projection, with the face represented Fibiger, '78; Kim et al., '76; Mehler, '71; ventromedially and the hindlimb dorsolateral-

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ly, the projection is diffuse (Rispal-Padel et al., '73; Strick, '73). Contraction of distal limb musculature was produced by stimulation of the ventrolateral half of VL, while axial and proximal limb musculature were activated by stimulation of the dorsomedial half of VL (Strick, '73). Cortical ablation does not eliminate such movements, probably because collaterals to the red nucleus from cerebello-thalamic fibers are activated (Asanuma and Hunsperger, '75). The EPN projection to thalamus includes the region which projects to cortical area 6 controlling proximal forelimb and trunk musculature, and to regions projecting to cortical areas 4 and 6 controlling facial musculature. The thalamic region projecting to the distal forelimb area of motor cortex, demonstrated with degeneration experiments in cats with VL lesions (Strick, '731, with electrophysiological experiments (Rispal-Padel et al., '731, and with horseradish peroxidase experiments with injections into monkey motor cortex (Strick, '761, does not receive an input from the EPN (figs. 2, 3) or the primate medial pallidum (Kim et al., '76). The major thalamic projection of the nucleus interpositus posterior of the feline cerebellum (Angaut, '70) is to the region of VL which projects to the arm area of motor cortex, and stimulation of the nucleus interpositus posterior evoked potentials primarily in the distal forelimb region of motor cortex (Massion and Rispal-Padel, '71). Therefore, the activity of distal, upper limb musculature may be modulated directly by the cerebellum, but not by the basal ganglia. There may be overlap in the projections to thalamus from the EPN and cerebellum for regions other than that related to the distal arm, however. The dentato-thalamic projection (Massion and Rispal-Padel, '71) appears to overlap with t h a t of the EPN. Unit studies provide inconclusive evidence about convergence of cerebellar and EPN outputs. Desiraju and Pupura ('67) found excitatory monosynaptic convergence on 20%of the thalamic cells, while Uno and Yoshida ('75) found less (8%)and it was of opposite sign (EPNevoked PSPs were inhibitory rather than excitatory). The EPN projection to CM terminated mostly in the rostral small celled part. The medial pallidal projection to the CM of primates is also restricted rostrally (Kim e t al., '76; Nauta and Mehler, '66). The small-celled portion of CM, which has different connec-

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tions than the rest of the nucleus, projects to the striatum and receives inputs from motor cortex but not spinothalamic inputs (Jones and Burton, '74; Mehler, '66). Projections to non-thalamic nuclei The density of silver grains was greater over the LHB than any other region following injections of amino acid into EPN. HRP experiments indicated that those cells which project to LHB are not unique, either in location or size; they were distributed throughout the nucleus, and the range, mean and distribution of cell sizes did not differ from that of other EPN cells. Herkenham and Nauta ('77) found rat EPN cells are distributed throughout the nucleus also. However, these authors reported that nearly all rat EPN cells were marked, whereas a smaller proportion of cat EPN cells was marked in this study in which the heaviest concentration of labeled cells was in the lateral hypothalamus. While EPN cells which project to LHB were distributed throughout the nucleus, these data suggest that rostral, but not caudal, EPN cells may project to LHB by way of stria medullaris. Nauta ('74)considered the stria medullaris and stria terminalis labeling to result from incorporation of amino acids by the EPN in cats. A preoptic projection by way of stria medullaris to LHB has been demonstrated in cats (Nauta, '58) and rats (Herkenham and Nauta, '77). In our experiments, only rostral injections of amino acid into EPN, in which the adjacent preoptic area or medial amygdala had uptake, resulted in measurable radioactivity in both stria medullaris and stria terminalis. Injection of HRP into stria terminalis did not result in labeling of EPN neurons. However, following HRP injections into stria medullaris, cells in rostral EPN were labeled. The pallido-habenular projection in r a t s (Herkenham and Nauta, '77) and monkeys (Kim et al., '76) is by way of the stria medullaris and does not enter the habenula ventrally. Golgi studies (Iwahori, '77) show that afferent fibers to the feline LHB enter the nucleus ventrally, as well as in the stria medullaris, and these autoradiographic and HRP studies suggest that most feline pallido-habenular fibers enter t h e LHB ventrally. Therefore, the course of feline pallido-habenular projections is more diverse than in rats or monkeys. Projections to t h e P P were found to arise from all portions of the EPN in both autoradi-

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ographic and HRP experiments. The axons were difficult to follow because of a low concentration of silver grains in autoradiographs. With small amino acid injections, only a fraction of the several hundred axons projecting there would be labeled. This, along with short survivals which show axons less than terminals, would contribute to the difficulty in following the path. Few HRP-containing cells were found following PP injections, relative to the LHB. This is in contrast to electrophysiological results (Larsen and Sutin, '78) which suggest that approximately equal percentages of cells project to LHB (7%)and PP (8%).The density of terminals in the vicinity of the injection may determine the amount of HRP uptake and subsequent visualization of cells (Jones and Leavitt, '74). Taken together the above results suggest that approximately equal numbers of cells project to LHB and P P but the terminal density is much greater in the former. The projection to P P in monkeys appears to be larger than in cats (Kim et al., '76; Kuo and Carpenter, '73; Nauta and Mehler, '66). The only information related to possible functional significance of this pathway is the observation that the terminal field overlaps with the mesencephalic locomotor region, i.e., that area which, when stimulated in mesencephalic cats, causes the animal to begin walking on a treadmill (Orlovsky and Shik, '76). Also, motor cortex, but not parietal or occipital cortex, projects to the primate PP (Kuypers and Lawrence, '67). In addition to projections to VA, CM, LHB, and PP, EPN may also project to STN and substantia nigra. Many fibers pass around the STN, so terminals there would be difficult to recognize with confidence. The previous reports of a medial pallidal input in primates to STN (Carpenter and Strominger, '67; Carpenter et al., '68) have been refuted (Kim et al., '761, and HRP injections into the feline substantia nigra (Grofova, '75; McBride and Larsen, '77) do not substantiate the projection. To recapitulate, labeled EPN cells were distributed throughout the nucleus regardless of the location of HRP injections in VA, LHB, or PP, and amino acids were transported to the same terminal fields regardless of the locations of the injections in EPN. This, along with the measurements of EPN somas showing that there is no difference in the size or shape of cells projecting to different nuclei,

leads to the conclusion that there is no anatomical distinction among the populations of EPN cells which project to different sites, either in morphology or location, although the number of EPN cells projecting to different nuclei differs. Electrophysiological studies on the projections of single EPN cells support this (Larsen and Sutin, '78). One must wonder how this organization, which lacks a point to point or topographical arrangement of projections, may relate to the function of the basal ganglia. The primate and feline cortico-striate (Kemp and Powell, '70; Webster, '65) and striato-pallidal (Niimi et al., '70; Szabo, '62; Voneida, '60) projections are topographically organized. This organization is loose since there is no somatotopic representation of pallidal units related to fore- and hindlimb movements (DeLong, '711, and individual EPN cells have convergent inputs from multiple sensory systems (Levine et al., '74). In view of the divergence of the projection from local parts of the EPN and convergence on VA from all parts of EPN, each region of the EPN may modulate movement about multiple joints and widespread regions of the body. Additionally, the non-thalamic projections of the EPN, which arise throughout the nucleus contiguous with the thalamic projections, may have motor functions: muscle spindle activity is modulated by stimulation of the habenula (Granit and Kaada, '52); striatal and pallidal conditioning stimuli modulate pyramidal tract evoked activity in tibialis anterior muscle of cats with motor cortex removed (Newton and Price, '75). LITERATURE CITED Angaut, P. 1970 The ascending projections of the nucleus interpositus posterior of the cat cerebellum: An experimental anatomical study using silver impregnation methods. Brain Res., 24: 377-394. Asanuma, H., and R. B. Hunsperger 1975 Functional significance of projection from the cerebellar nuclei to the motor cortex in the cat. Brain Res., 98; 73-92. Carpenter, M. B., J. W. Corell and A. Hinman 1960 Spinal tracts mediating subthalamic hyperkinesia. Physiologic effects of selective partial cordotomies upon dyskinesia in Rhesus monkey. J. Neurophysiol., 23: 288-304. Carpenter, M. B., R. A. R. Fraser and J. E. Shriver 1968 The organization of pallidosubthalamic fibers in the monkey. Brain Res., 11: 522-559. Carpenter, M. B., and N. L. Strominger 1967 Efferent fibers of the subthalamic nucleus in the monkey. A comparison of the subthalamic nucleus, substantia nigra, and globus pallidus. Am. J. Anat., 121: 41-72. Carter, D. A., and H. C. Fibiger 1978 The projections of the entopeduncular nucleus and globus pallidus in rat as demonstrated by autoradiography and horseradish peroxidase histochemistry. J. Comp. Neur., 177: 113-124.

ENTOPEDUNCULAR NUCLEUS PROJECTIONS Conrad, L. C. A., C. M. Leonard and D. W. Pfaff 1974 Connections of the median and dorsal raphe nuclei in t h e rat: An autoradiographic and degeneration study. J. Comp. Neur., 256: 179-200. Conrad, L. C. A,, and D. W. Pfaff 1976 Efferent8 from medial basal forebrain and hypothalamus in the rat. J. Comp. Neur., 269: 185-220. Cowan, N. W., D. I. Gottlieb, A. E. Hendrickson, J. C. Price and T. A. Woolsey 1972 The autoradiographic demonstration of axonal connections in t h e central nervous system. Brain Res., 37: 21-51. DeLong, M. R. 1971 Activity of pallidal neurons during movement. J. Neurophysiol., 34: 414-437. Denny-Brown, D. 1962 The Basal Ganglia. Oxford Univ. Press, Amen House, London, 144 pp. Desiraju, T., and D. P. Purpura 1967 Synaptic convergence of cerebellar and lenticular projections to the thalamus. Brain Res., 25: 544-547. Edwards, S. B. 1975 Autoradiographic studies of the projections of the midbrain reticular formation: Descending projections of nucleus cuneiformis. J. Comp. Neur., 162: 341-358. Fox, C. A,, D. E. Hillman, K. A. Siegsmund and L. A. Sether 1966 The primate globus pallidus and its feline and avian homologues: A Golgi and electron microscope study. In: Evolution of the Forebrain. R. Hassler and H. Stephan, eds. Thieme, Stuttgart, pp. 237-248. Frigyesi, T. L., and J. Machek 1970 Basal ganglia-diencephalon synaptic relations in the cat. I. An intracellular study of dorsal thalamic neurons during capsular and basal ganglia stimulation. Brain Res., 20: 201-217. Granit, R., and B. R. Kaada 1952 Influence of stimulation of central nervous structures on muscle spindles in cat. Acta Physiol. Scand., 27: 130-160. Graybiel, A. M. 1975 Wallerian degeneration and anterograde tracer methods. In: The Use of Axonal Transport for Studies of Neuronal Connectivity. W. M. Cowan and M. Cuenod, eds. Elsevier, Amsterdam, pp. 47-68. Grofova, I. 1975 The identification of striatal and pallidal neurons projecting to substantia nigra. An experimental study by means of retrograde axonal transport of horseradish peroxidase. Brain Res., 91: 286-291. Grofova, I., and E. Rinvik 1974 Cortical andpallidal projections to the nucleus ventralis lateralis thalami. Anat. Embryol., 246: 113-132. Hardy, H., and L. Heimer 1977 A safer and more sensitive substitute for diaminobenzidine in the light microscopic demonstration of retrograde and anterograde axonal transport of HRP. Neurosci. Letters, 5: 235-240. Herkenham, M., and W. J. H. Nauta 1977 Afferent connections of the habenular nuclei in t h e rat. A horseradish peroxidase study with a note on the fiber of passage problem. J. Comp. Neur., 2 73: 123-146. Hore, J., J. Meyer-Lohman and V. B. Brooks 1977 Basal ganglia cooling disables learned arm movements of monkeys in the absence of visual guidance. Science, 295: 584-586. Iwahori, N. 1977 A Golgi study on the habenular nucleus of t h e cat. J. Comp. Neur., 271: 319-344. Jasper, H. H., and C. Ajmone-Marsan 1954 A Stereotaxic Atlas of the Diencephalon of the Cat. National Research Council of Canada, Ottawa. Johnson, T. N., and C. D. Clemente 1959 An experimental study of fiber connections between the putamen, globus pallidus, ventral thalamus, and midbrain tegmentum in the cat. J. Comp. Neur., 223: 83-101. Jones, E. G., and H. Burton 1974 Cytoarchitecture and somatic sensory connectivity of thalamic nuclei other than the ventrobasal complex in the cat. J. Comp. Neur., 154: 395-432.

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Jones, E. G., and R. Y. Leavitt 1974 Retrograde axonal transport and the demonstration of non-specific projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat and monkey. J . Comp. Neur., 254: 349-378. Kaelber, W. W. 1970 An experimental study of subcortical projections of the nucleus ventralis anterior in the cat. J. Anat., 207: 399-406. Kemp, J. M., and T. P. S. Powell 1970 The cortico-striate projection in the monkey. Brain, 93: 525-546. Kim, R., K. Nakano, A. Jayaraman and M. B. Carpenter 1976 Projections of the globus pallidus and adjacent structures: An autoradiographic study in t h e monkey. J. Comp. Neur., 269: 263-289. Konigsmark, B. W., B. W. Kalyanaraman, P. Corey and E. A. Murphy 1969 An evaluation of techniques in neuronal population estimates: The sixth nerve nucleus. The Johns Hopkins Medical Journal, 225: 146-158. Kuo, J. S., and M. B. Carpenter 1973 Organization of pallidothalamic projections in the rhesus monkey. J. Comp. Neur., 252: 201-236. Kuypers, H. G. J. M., and D. G. Lawrence 1967 Cortical projections to the red nucleus and the brainstem in the rhesus monkey. Brain Res., 4: 151-188. Larsen, K. D., and J. Sutin 1978 Output organization of the feline entopeduncular and subthalamic nuclei. Brain Res., 257: 21-31. Lasek, R. 1968 Axoplasmic transport in the cat dorsal root ganglion cells: As studied with [ W-L-leucine.Brain Res., 7: 360-377. Levine, M. S., C. D. Hull and N. A. Buchwald 1974 Pallidal and entopeduncular intracellular responses to striatal, cortical, thalamic and sensory inputs. Exp. Neurol., 44: 448-460. Massion, J., and L. Rispal-Padel 1971 Spatial organization of the cerebello-thalamo-corticalpathway. Brain Res., 40: 61-65. McBride, R. L., and K. D. Larsen 1977 Descending projections of the feline globus pallidus. Neurosci. Abstr., 3: 42. McBride, R., and J. Sutin 1977 Amygdaloid and pontine projections to the ventromedial nucleus of t h e hypothalamus. J. Comp. Neur., 274: 377-396. Mehler, W. R. 1966 Further notes on the center median nucleus of Luys. In: The Thalamus. D. P. Purpura and M. D. Yahr, eds. Columbia University Press, New York, pp. 109-128. 1971 Idea of a new anatomy of the thalamus. J. Psychiat. Res., 8: 203-217. Nauta, H. J. W. 1974 Evidence of a pallidohabenular pathway in the cat. J. Comp. Neur., 256: 19-28. Nauta, W. J. H. 1958 Hippocampal projections and related neural pathways to the midbrain in the cat. Brain, 82: 319-340. Nauta, W. J. H., and W. J. Mehler 1966 Projections of the lentiform nucleus in the monkey. Brain Res., 2: 3-42. Newton, R. A,, and D. L. Price 1975 Modulation of cortical and pyramidal tract induced motor responses by electrical stimulation of the basal ganglia. Brain Res., 85: 403-422. Niimi, K., T. Ikeda, S. Kawamura and M. Inoshita 1970 Efferent projections of the head of the caudate nucleus in the cat. Brain Res., 21: 327-343. Orlovsky, G. M., and M. L. Shik 1976 Control of locomotion: A neurophysiological analysis of the cat locomotor system. In: International Review of Physiology. Neurophysiology 11. R. Porter, ed. University Park Press, Baltimore, Vol. 10, pp. 281-317. Papez, J. W. 1940 A summary of fiber connections of the basal ganglia with each other and with other portions of the brain. A. Res. Nerve and Ment. Dis. Proc., 21: 21-68.

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Ranson, S. W., S. W. Ranson, J r . and M. Ranson 1941 Fiber connections of the corpus striatum as seen in Marchi preparations, Arch. Neurol. Psychiat., 46: 230-249. Rinvik, E. 1968 A re-evaluation of the cytoarchitecture of the ventral nuclear complex of the cat's thalamus on the basis of corticothalamic connections. Brain Res., 8: 237-254. Rinvik, E., and I. Grofova 1974 Light and electron microscopical studies of the normal nuclei ventralis lateralis and ventralis anterior thalami in the cat. Anat. Embryol., 146: 57-93. Rispal-Padel, L., J. Massion and A. Granzetto 1973 Relations between the ventrolateral thalamic nucleus and the motor cortex and their possible role in the cerebral organization of motor control. Brain Res., 60: 1-20. Strick, P. L. 1973 Light microscopic analysis of the cortical projection of the thalamic ventrolateral nucleus in the cat. Brain Res., 55: 1-24. 1976 Anatomical analysis of the ventrolateral thalamic input to primate motor cortex. J. Neurophysiol., 39: 1020-1031. Sutin, J., and R. L. McBride 1977 Anatomical analysis of neuronal connectivity. In: Methods in Psychobiology: Advanced Laboratory Techniques in Neuropsychology

and Neurobiology. R. D. Myers, ed. Academic Press, New York, Vol. 111, pp. 1-26. Swanson, L. W., W. M. Cowan and E. G. Jones 1974 An autoradiographic study of the efferent connections of the ventral lateral geniculate nucleus in the albino r a t and the cat. J. Comp. Neur., 156: 143-164. Szabo, J. 1962 Topical distribution of the striatal efferents in the monkey. Exp. Neurol., 5; 21-38. Uno, M., and M. Yoshida 1975 Monosynaptic inhibitions of thalamic neurons produced by stimulation of the pallidal nucleus in cats. Brain Res., 99; 377-380. Voneida, T. J. 1960 An experimental study of the course and distribution of fibers arising in t h e head of the caudate nucleus of the cat and monkey. J. a m p . Neur., 115: 75-87. Webster, K. E. 1965 The cortico-striate projection in the cat. J. Anat., 99: 329-337. Wilson, S.A. K. 1914 An experimental research into the anatomy and physiology of the corpus striatum. Brain, 36; 427-492. Woodburne, R. T., E. C. Crosby and R. E. McCotter 1946 The mammalian midbrain and isthmus regions. Part 111. A. The relations of the tegmentum of the midbrain with the basal ganglia in Macaca mdatta. J. Comp. Neur., 86: 67-92.

Note added in proof' The branching pattern of EPN axons was recently investigated by antidromic activation from VA-VL, CM, PP, and LHB (M. Filion and C. Harnois 1978. A comparison of projections of entopeduncular neurons to the thalamus, the midbrain, and the habenula in the cat. J. Comp. Neur., 181: 763-7801, The absolute percentages of cells which they reported projecting to VA-VL (68%)and LHB (25%) is approximately two to three times that reported by us (with retrograde HRP transport) or by Larsen and Sutin ('68). However, this difference is largely a t tributable to the method in which the percentages were calculated, since Filion and Harnois reported the number of EPN cells activated from each site as a percentage of the total number of activated EPN cells and omitted those which were isolated but not activated. In spite of the differences among studies in absolute percentages, cells identified as projecting to VA-VL were three times as common as those projecting to LHB in each study. While we consider the estimates of the percentages of cells projecting to the terminal fields in VA-VL and LHB to be consistent among the different reports, we think Filion and Harnois have seriously overestimated the projection to CM (69%)by activating fibers of passage. Their illustrated CM-electrode placement is within 2 mm of the thalamic fasciculus, in which fibers course to VA-VL, and both their estimate and readily available data show that their 1.5 mA stimulus current effectively spreads this distance. In no case did Filion and Harnois interact stimuli a t two sites to produce a collision and verify by measuring the conduction time that a collateral instead of a n axon passage was activated. Furthermore, their estimate is inconsistent with our anatomical data and the electrophysiological data of Larsen and Sutin ('78) which suggest that the projection to CM is much smaller (in number of cells, terminal density, and size of terminal field) than to VA-VL. Equally inconsistent is their report of a large (54%)projection to PP, but we cannot resolve this discrepancy.