Efferent Connections of the Caudate Nucleus in the Virginia Opossum J O H N PARKER MICKLE Depcirtmeiat o f Ps?jchology, Massachusetts Institute of Technology, C(iJJLbI-ldge, Mmscichusetts, u. s. A.
ABSTRACT The efferents of the opossum‘s caudate nucleus were investigated by charting the fiber degeneiations produced by electrolytic lesions in various parts of this nucleus. By the aid of a modified Fink-Heimer procedure, degenerating fibers were traced from each of the lesions to the small globus pallidus in which they appeared to be distributed in an orderly dorsoventral pattern. Fibers from all lesion sites in the caudate nucleus were found to terminate in the entopeduncular nucleus. In the substantia nigra, caudatofugal-fiber degeneration was confined in all cases to the rostra1 part of the pars reticulata, and was densest in the medial one-half of this nucleus. Only from lesions in the ventromedial part of the caudate nucleus could degenerating fibers be traced to the nucleus ansae lenticularis. N o fiber degeneration could be traced rostrally from the lesions, or to the putamen, red nucleus, subthalamic nucleus, or pons.
Knowledge of the efferent connections of the mammalian striatum dates back to the time around 1900 when Bechterew ( l 8 9 9 ) , Probst ( ’ 0 3 ) , and Griinstein (’11) demonstrated the fine-fibered projection of the putamen and caudate nucleus to the globus pallidus, a connection that was repeatedly confirmed in later experimental studies in a variety of mammalian species. Striatal efferents continuing beyond the globus pallidus to the substantia nigra were first reported by Edinger (’ll), and later were confirmed by Riese (’24a,b), Rundles and Papez ( ’ 3 7 ) , Voneida ( ’ 6 0 ) , Szabo (’62) and others. Since striatal efferents have been consistently traced to the globus pallidus in a variety of mammalian species, it seemed reasonable to expect that an experimental study of the caudatal projections would help to identify the cell group or cell groups that compose the globus pallidus in which the pallidum has not yet been delineated with certainty. Prominent among such species is the opossum. The cytoarchitecture of the opossum’s corpus striatum has been the subject of two recent publications (Martin and Hamel, ’67; Oswaldo-Cruz and RochaMiranda, ’68), in both of which the label, globus pallidus, is restricted to a remarkably small and apparently undivided cell J.
group that resembles in its structure the external segment of the primate globus pallidus ( Oswaldo-Cruz and Rocha-Miranda, ’68). The entopeduncular nucleus, widely regarded as the non-primate homologue of the primate’s internal pallidal segment (Fox and Schmitz, ’44; Martin and Hamel, ’67; Nauta and Mehler, ’61) has not been identified with certainty in the opossum, judging from the differing anatomical descriptions given by various authors (Loo, ’31; Martin and Hamel, ’67; Oswaldo-Cruz and Rocha-Miranda, ’68). Loo (’31) describes several cell groups closely associated with the opossum’s globus pallidus : a nucleus ansae lenticularis ventral and medial to the globus pallidus, and two cell aggregates, one of which lies embedded in the cerebral peduncle (“intrapeduncular nucleus”), the other immediately dorsal to the peduncle (“suprapeduncular nucleus”). A major purpose of the present study was, to determine which, if any, of these cell groups might receive striatofugal fibers, and hence, be considered part of the opossum’s globus pallidus. Anatomically the caudate nucleus of the opossum lends itself well to an experimental demonstration of its efferents by its relatively large size and the sparseness of interspersed fibers of the internal capsule. Such experimental studies are also favored 3 73
JOHN PARKER MICKLE
by the absence of a plate-like corpus callosum and the small volume of the opossum's neocortex. MATERIALS AND METHODS
The experiments described below were performed on 25 young adult male and female opossums (Didelphis virginiana) weighing from 1 to 2 kg. In each of these animals a single electrolytic lesion was placed either in the caudate nucleus or in a brain structure that unavoidably would be pierced by the electrode in its passage to the caudate nucleus. All lesions were made by the aid of a small dissecting pin insulated except for the tip, mounted on a standard stereotaxic carrier, and connected to a constant-current stimulator. The indifferent electrode was connected to the animal's tail. Each animal was deeply anesthetized by intraperitoneal injection of pentobarbital (30 mg per kg) and placed in a standard Horsely-Clarke stereotaxic instrument. Stereotaxic coordinates for all lesions were obtained from Oswaldo-Cruz and RochaMiranda's ('68) atlas. The lesions were made by passing a direct current of 1 mA for eight seconds. In each case, before producing the lesion, a current of 0.5 mA was delivered to the target area in order to observe any motor responses that might result. After survival times ranging between 4 and 14 days, the animals were deeply anesthetized with pentobarbital and perfused through the left cardiac ventricle with 250 ml of normal saline followed by 500 ml of 10% formalin in saline. The brain was removed, stored in 10% formalin for one to two weeks, transferred to a 10% sugarformalin solution for three days, embedded in albumin, and sectioned in either the sagittal or coronal plane on a freezing microtome at 30 thickness. Of each brain, every fifth section was stained for cells with cresylechtviolet, and an adjacent section by a modified Fink-Heimer ('67) silver impregnation for degenerating axons and terminals. In the latter procedure it was found advantageous to lower the silver nitrate concentration to 0. l % in solution A, and to omit solution B altogether. Further reduction in silver concentration was found to result in impregnation of normal
synaptic terminals, especially in the brain stem. To record the microscopic observations, an enlarged projection image of a silver-stained section was traced onto paper, outlining the major fiber tracts and cell groups. Degeneration was then indicated on these tracings from microscopic study, using blood vessels, recognizable cell groups and fiber tracts as landmarks. A matched section stained for cells was always available for cytoarchitectural identification and delineation of the lesion. EXPERIMENTAL FINDINGS
The intraoperative stimulation of the caudate nucleus through the in-dwelling electrode invariably produced a twitch of the opossum's tail toward the stimulated side. In all cases in which other motor responses were elicited the lesion was found to be in the internal capsule. No postoperative wound infections were encountered. For the purpose of the present study the optimal survival time was found to be seven days. Cases of shorter survival often showed inconclusive pictures of axon degeneration, whereas longer survival times led to a reduction in the impregnation of degenerating fine fibers and axon terminals Included in the present material are six cases of lesion restricted to the caudate nucleus (fig. 1). Two of these lesions were produced by a vertically oriented electrode, and the remainder through the contralateral cortex 60-70" from the vertical. The composite sagittal extent of these lesions is shown in figure 2. This diagram was constructed on regular graph paper, using the coordinates of the caudate nucleus and anterior commissure given by Oswaldo-Cruz and Rocha-Miranda ('68) ; the lesions have been drawn into the figure on the basis of an approximation of their extents as judged microscopically from anatomical landmarks. Control material consisted of two cases of partial decortication over the area penetrated by the vertically inserted electrodes, and three cases of electrolytic lesion of the hippocampus and fornix placed through the contralateral cortex. These controls were necessary to determine the fiber degeneration caused by the electrode tracts. Figures 3-5 are composed of serial semi-
CAUDATE E F F E R E N T S I N THE OPOSSUM CAUDATE
Fig. 1 This figure represents semi-diagrams of the caudate lesions reported. The first two lesions were made with a vertically oriented probe, and the remainder were made via a contralateral approach with the probe 60-70" from the vertical.
diagrams of the degeneration patterns charted in three of the six cases here reported. Opossum V l l (fig. 3 ) presents a large electrolytic lesion restricted to the dorsomedial area of the caudate nucleus. It was placed by the aid of an electrode introduced vertically through the ipsilateral cortex. Slender bundles of densely packed, h e degenerating fibers are readily traced from the lesion through the anterior limb of the internal capsule and anterior putamen (figs. 3A,B, s),but there is no evidence that any of these caudatofugal fibers terminate in the putamen. The most lateral of these degenerating fibers enter the globus pallidus from the dorsal side, and become lost among degenerating axon terminals that occupy the entire extent of this small cell region and are particularly numerous near its dorsal surface (fig. 3 C ) . Many other caudatofugal fibers, instead of entering the globus pallidus, pass caudally in a middle stratum of the internal
capsule. A moderate number of these degenerating fibers, retaining their midposition in the cerebral peduncle, terminate in the small entopeduncular nucleus (figs. 3E, 7 ) . The remaining fibers of this intracapsular group continue caudally, then turn dorsally to enter the rostral part of the substantia nigra in which they are lost amid a terminal degeneration that is confined to the pars reticulata and is densest near the medial border of the nucleus (figs. 3G, 8). No degenerating caudatal efferents could be traced distal to this level. The lesion in Opossum XVI (figs 1, 2 ) is somewhat similar to that in VII, but it was made through the contralateral cortex and ipsilateral hippocampus, and is located more caudally in the caudate nucleus. The degeneration pattern in this case corresponds cIosely to that in VII, except that the degenerating fibers entering the globus pallidus do so more medially and more ventrally. Opossums IV a n d X X I I (fig. 1 ) had
JOHN PARKER MICKLE LESIONS
SAGITTAL VI E
0 M M I S S U RE
Fig. 2 The composite sagittal extent of the six lesions reported are represented i n this diagram. This was constructed on regular graph paper using the co-ordinates from the atlas by Oswaldo-Cruz and Rocha-Miranda ('68). The lesions have been drawn onto the graph 011 the basis of an approximation of their extents as judged microscopically from anatomical landmarks.
lesions located more medially and ventrally than the two previously discussed. I n both cases, degenerating caudatofugal fibers entered the globus pallidus through its ventroinedial border. Otherwise the degeneration patterns in these two cases were indistinguishable from those on opossums VII and XVI. O p o s s u m XXV (fig. 4 ) had a small lesion that was located quite far rostrally and ventrally and involved largely the cytoarchitecturally distinct rostral area of the opossum's caudate nucleus described by
Martin and Hamel ('67). Degenerating fibers in this case passed from the lesion through and under the anterior commissure (fig. 4 B ) . A considerable number of these fibers appeared to terminate ventral and medial to the caudal pole of the globus pallidus in a cell region described by Loo ('31) as the nucleus ansae lenticularis (fig. 4 C ) . As pointed out by Oswaldo-Cruz and Rocha-Miranda ('68) this nucleus is only vaguely delimited from the globus pallidus and can be distinguished from the latter only by the fact that it contains,
Abbreuiutimzs Ansa lenticularis ic, Inlerior colliculus Anterior c o i ~ l i i ~ i s s ~ r e I v , Lateral ventricle Caudate nucleus nid. Mediodorsal nucleus External capsule pd, Peduncle Internal capsule p u , Pu tainen Claustrum r n , Red nucleus cs, Superior calliculus ep, ~ ~nucleus si, ~ Substantia innoininata ~ sn, Substantia nigra gm, Medial geniculate gp, Globus pallidus st, Subthalamic nucleus al, ca, cd, ce, ci, cl,
Fig. 3 This serial semi-diagram demonstrates the degeneration obtained from a large dorsomedial lesion in the caudate nucleus of opossum VII. Fibers pass into the internal capsule and through the putamen ( A ) . The most lateral of these degenerating fibers enter the dorsal part o f the globus pallidus and terminate diffusely in this nucleus ( C ) . More niedially located fibers pass caudally with a moderate number terminating in the small entopeduncular nucleus ( E ) . The remaining caudatofugal fibers pass caudally then turn dorsally to terminate in the medial part of the substantia nigra, pars reticulata ( G ) .
Axonal Degen. Terminal Degen.
Axonal D e g e n .
CAUDATE EFFERENTS IN T H E OPOSSUM
among neurons similar to those of the also suggest that it is topographically orglobus pallidus, larger cells with a coarser ganized in the dorsoventral axis in the Nissl substance. No degenerating fibers sense that the caudodorsal-to-rostroventral could be traced to the globus pallidus dimension of the caudate nucleus appears proper in this case, but the findings in the to correspond to a dorsoventral gradient in entopeduncular nucleus and substantia the terminal distribution of the caudatopalnigra closely resemble those in the two lidal projection. The rostroventral region foregoing cases. A small number of degen- of the caudate nucleus immediately bordererating fibers in the internal capsule were ing the dorsum of the anterior commissure traced medially and dorsally into the even appears to project to a cell group mediodorsal and paracentral nuclei of the which has been described as lying ventral to the globus pallidus proper : Loo's ( ' 31) thalamus (fig. 4E,F). Figure 5 represents the sagitally cut nucleus ansae lenticularis. The findings in brain of opossum XXIV. The lesion in this the present case XXV clearly identify this case was placed very far anterodorsally. cell group as a recipient of caudatofugal The relationship of the degenerating fibers fibers, and this would suggest that the nuto the anterior comrnissure is more readily cleus is actually a component of the globus appreciated in this case than in transverse pallidus with which, moreover, i t shares series, and their termination in the globus some cytological features. All caudatofugal pallidus and entopeduncular nuclei is fibers are thin, and reach the pallidum by coursing in slender, compact fascicles easily recognized (figs. 5F, 9 ) . Control cases. In the cases of dorsal through the putamen, internal capsule or neocortex lesions abundant degenerating anterior commissure, depending on their fibers could be traced to the caudate nu- site of origin in the caudate nucleus. Fibers cleus and thalamus, but no fiber degenera- from the most ventral and anterior part tion appeared in the globus pallidus, ento- of the nucleus tend to reach the pallidum peduncular nucleus or substantia nigra. by passing either through or under the Control lesions in the hippocampus in anterior commissure. Fibers originating in three animals likewise failed to elicit fiber more dorsal regions stream into the indegeneration in the cell regions receiving ternal capsule above the anterior commissure to reach the globus pallidus. caudatofugal fibers. All the caudate lesions in this study elicDISCUSSION ited fiber degeneration in the small cell Caudatopallidal fibers. From a study group labeled "intrapeduncular nucleus" by by the Marchi method in the cat, Ranson Loo ' 3 1 ) . Both this observation and the et al. ('41) concluded that the caudate nu- position of the nucleus in the dorsomedial cleus projects primarily to the external seg- part of the cerebral peduncle just rostra1 to ment of the globus pallidus. Mettler ('45) the subthalamic nucleus suggests that this confirmed this projection in the baboon cell group indeed corresponds to the enand monkey and specified that the projec- topeduncular nucleus of other mammalian tion involved in particular the dorsal half forms, as already assumed by Oswaldoof the external pallidal segment. Cowan Cruz and Rocha-Miranda ('68). As pointed and Powell ('66) noted a mediolateral out by Fox and Schmitz ('44) and Nauta topography in the caudatopallidal system and Mehler ('61) this nucleus of nonin the monkey. Szabo ('62, '70) and John- primate mammals appears to be the homoson and Rosvold ('71) in the monkey, and logue of the internal division of the priVoneida ('60) and Niimi et al. ('70) in the mate globus pallidus. Niimi et al. ('70) cat found evidence of a n orderly medio- found that the entopeduncular nucleus in lateral and anteroposterior topography of the cat receives fibers exclusively from the the caudatofugal fibers to both the globus dorsolateral part of the caudate nucleus, but the present findings indicate that the pallidus and the substantia nigra. In the opossum, the caudatofugal pro- projection in the opossum arises from a jection to the opossum's small globus pal- much wider area of the caudate nucleus. On the basis of the present findings, lidus appears to be diffuse in the anteroposterior direction, but the present findings the opossum's nucleus of the ansa lenticu-
CAUDATE E F F E R E N T S I N THE OPOSSUM
laris could be interpreted as a further component of the pallidum. However, both its location very near the globus pallidus (i.e. the external segment of the pallidum), and the fact that - contrary to the entopeduncular nucleus - it appears to receive caudatofugal fibers from only a restricted rostroventral region of the caudate nucleus, suggest that this small cell group is a component of the external rather than the internal segment of the pallidum. Cazidatothalmic fibers. Shulman and Auer ('57) reported evidence of a projection from the caudate nucleus to the centrum medianum and the parafascicular nucleus in the thalamus of the monkey. Voneida ('60) Szabo ('62, '70),Niimi et al. ('70) and Johnson and Rosvold ('71) could not confirm such a caudatothalamic connection in the cat, and Nauta and Mehler ('66) failed to identify i t in the monkey, although they did demonstrate a projection to the centrum medianum from the internal segment of the globus pallidus. In the present study, the lesion in only one case (XXV) caused degeneration of fibers to the thalamus, in particular the mediodorsal and paracentral nuclei. Since this lesion may have interrupted some corticofugal fibers in the medial margin of the internal capsule (fig. l ) , it seems possible that this thalamopetal-fiber degener ation represents a corticothalamic rather than a caudatothalamic connection. Johnson ( '61 ) reported evidence of a caudatosubthalamic projection in the cat, but subsequent studies (Voneida ('601, Szabo ('62, '70), Niimi et al. ('70)) have failed to confirm this finding. In the present study likewise, no direct caudatosubthalamic connections could be demonstr ated. C a u d a t o f u g a l fibers in the medial forebrain bundle. From a study of normal opossum material Loo ('31 ) reported fibers from the ventromedial region of the caudate nucleus that join the medial forebrain bundle. No evidence of such fibers appeared in the present study, but it must be noted that none of the lesions here studied involved regions of the caudate nucleus ventral to the level of the anterior commisure. Cazidatonigral fibers. Papez ( ' 3 8 ) was
the first to propose a striatonigral connection in the monkey. Vogt and Vogt ('37) had suggested this connection in man in 1937. In a study by the Marchi method, Ranson et al. ('41) could trace no caudatofugal fibers beyond the globus pallidus in the monkey. Voneida ('60) and Johnson ('61), using the Nauta method, were able to demonstrate a projection to the rostromedial part of the substantia nigra pars reticulata in the cat. Subsequently Szabo ('62, '70), Niimi et 31. ('70) and Johnson and Rosvold ('71) defined a mediolateral and anteroposterior organization in the caudatonigral projection. The present findings have revealed a projection from the caudate nucleus to the rostra1 part of the substantia nigra pars reticulata, most massively distributed to the medial half of this region. All caudate lesions elicited degeneration in this area, and no evidence of a inediolateral or anteroposterior topography in the caudatonigral connections was found. No evidence of striatal projections to the red nucleus or the pontine nuclei (Wilson, '14) appeared in this study. SUMMARY
The efferent connections of the caudate nucleus were studied in 25 opossums by the aid of a modified Fink-Heimer silver stain. The study revealed a dorsoventrally organized projection to the globus pallidus; and additional caudatofugal projections to the entopeduncular nucleus and the rostromedial part of the substantia nigra, pars reticulata. The cytoarchitecturally distinct rostroventral part of the caudate nucleus was found to project to the large-celled nucleus ansae lenticularis. No evidence of a direct caudatothalamic connection appeared, and no caudatofugal fibers could be traced caudally beyond the substantia nigra. ACKNOWLEDGMENTS
My warmest appreciation is extended to Dr. W. J. H. Nauta for his patience and cooperation during the preparation of this report. Also, the work could not have been completed without the technical assistance of Miss D. Majors and Mrs. D. Pong. This project was supported in part by the PHS Grant 5-T01-NS05519-06.
JOHN PARKER MICKLE LITERATURE CITED
Bechterew, W. van 1889 Die Leitungsbahnen im Gehirn und Riickenmark. Georgi, Leipzig. Cown, W. M., and T. P. S . Powell 1966 Striopallidal projections in the monkey. J. Neural., Neurosurg. and Psychiat., 20: 426-439. Edinger, L. 1911 Vorlesungen uber den Bau der nervosen Zentralorgane. Eighth ed. Val. I . Vogel, Leipzig. Fink, R. P., and L. Heimer 1967 Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Res., 4: 369-374. Fox, C. A., and J. T. Schmitz 1944 The substantia nigra and the entopeduncular nucleus in the cat. J. Comp. Neur., 80: 323-334. Griinstein, A. M. 1911 Zur Frage von den Leitungsbahnen des Corpus Striatum. Neurol. Zbl., 30: 659-665. Johnson, T. N. 1961 Fiber connections between the dorsal thalamus and the corpus striatum in the cat. Exp. Neural., 3 : 556-569. Johnson, T. N., and H. E. Rosvold 1971 Topographic projections on the globus pallidus and the substantia nigra of selectively placed lesions in the precommissural caudate nucleus and putamen in the monkey. Exp. Neural., 33: 584-596. Loo, Y . T. 1931 The forebrain of the opossum DidelDhvs -~ vireiniana. Part 11. Histolorrv. -. J . Comp. Neur., 52: 1-148. Martin, G. F., and E. G. Hamel 1967 The striatum of the opossum (Didelphis virginiana). J . Comp. Neur., 131: 491-516. Metrler, F. A. 1945 Fiber connections of the corpus striatum of the monkey and baboon. J . Comp. Neur., 82: 169-204. Nauta, W. J . H., and W. R. Mehler 1961 Some efferent connections of the lentiform nucleus in the monkey and cat. Anat. Rec., 139: 260. I966 Projections of the lentiform n u cleus in the monkey. Brain Research, I : 3-42. Niimi, K., T. Ikeda, S . Kawamura and H. Inoshita 1970 Efferent projections of the head of the caudate nucleus in the cat. Brain Research, 21: 327-344. Oswaldo-Cruz, E., and C. E. Rocha-NIiranda 1968
The Brain of the Opossum ( D i d e l p h i s mnrstcpicclis ). Instituto de Biofisico Universidade Federal do Ria de Janeiro, Ria de Janeiro, Brasil. Papez, J. W. 1938 Reciprocal connections of the striatum and pallidum i n the brain of Pithecus ( M a c a w s ) rhesus. J . Comp. Neur.. 69: 329-350. Probst, M. 1903 Ueber die Rinden-Sehhugelfasern des Riechfeldes, iiber das Gewolbe, die Zwinge, die Randbogenfasern, iiber die Schweifenkernfaserung, und iiber die Vertheilung der Pyramidenfasern im Pyranlidenareal. Arch. Anat. Physiol., Anat. Abth., H IZ-V: 138-152. Ranson, S. W., S. W. Ranson, Jr. and M. Ranson 1941 Fiber connections of the corpus striatuin a s seen in Marchi preparations. Arch. Neural. Psychiat., 46: 230-249. Riese, W . 1924a Zur vergleichenden Anatomie der striofugalen Faserung. Anat. Anz., 57: 487-494. 1924b Beitrage zur Faseranatomie der Stammganglien. J. Psychol. Neural., 31: 81-122. Rundles, R. W., and J . W. Papez 1937 Connections between the striatum and substantia nigra in a human brain. Arch. Neurol. Psychiat., 38: 550-563. Shulman, E., and J. Auer 1957 Caudate efferents to the thalamus. Anat. Rec., 127: 363. Szabo, J. 1962 Topical distribution of the striatal efferents in the monkey. Exp. Neural., 5: 21-36. ___ 1970 Projections from the body of the caudate nucleus in the Rhesus monkey. Exp. Neural., 27: 1-15. Vogt, C., and 0. Vogt 1937 Sitz und Wesen der Krankheiten im Lichte der topistischen Hirnforschung und des Variierens der Tiere. I. Befunde der topistischen Hirnforschung als Beitrag zur Lehre vom Krankheitssitz. J . Psychol. Neural. (Leipzig), 47: 237-457. Voneida, T. 1960 An experimental study of the course and destination of fibers arising in the head of the caudate nucleus in the cat and monkey. J . Comp. Neur., 115: 75-88. Wilson, S . A . K. 1914 An experimental research into the anatomy and physiology of the corpus striatum. Brain, 36: 427-492.
The entopeduncular nucleus in this photomicrograph from opossum XXIV is filled with degenerating terminals. A fascicle of degenerating fibers is seen entering the nucleus to the left. x 360.
This photomicrograph of the substantia nigra, pars reticulata from opossum VII shows the dense terminal degeneration found in this area from a lesion i n the caudate nucleus. The degeneration is most dense i n the medial part of this nucleus. x 360.
This photomicrograph shows the extensive degeneration in the entopeduncular nucleus of opossum VII. A fascicle in the cerebral pedunde with degeneratmg fibers of passage is seen to the right. :i360.
Fine degenerating fibers from the caudate nucleus in the internal capsule are well seen in this photomicrograph from opossum VII. The caudate nucleus is located to the left. x 200.
E X P L A N A T I O N O F FIGURES
CAUDATE EFFEREPJTS IN THE OPOSSUM John Parker Mickle