THE JOURNAL OF COMPARATIVE NEUROLOGY 306585-601 (1991)

Organization of Midbrain Catecholamine-Containing Nuclei and Their Projections to the Striatum in the North American Opossum, Didelphis uirginiana JAMES C. HAZLETT, RAYMOND H. HO, AND GEORGE F. MARTIN Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201 (J.C.H.) and Department of Anatomy, The Ohio State University College of Medicine, Columbus, Ohio 43210 (R.H.H., G.F.M.)

ABSTRACT Presumptive catecholamine (CAI neurons in the opossum midbrain were identified by tyrosine hydroxylase immunohistochemistry.In the midline, small to moderate numbers of CA cells were present in the rostral third of the nucleus raphe dorsalis and throughout the nucleus linearis. Ventrolaterally, such cells were observed in the deep tegmental reticular formation, in all subnuclei of the ventral tegmental area, and in the three subdivisions of the substantia nigra. The CA cells in these areas conform to the dopamine cell groups, AS, A9, and A10 as described in the rat. In several areas there appeared to be no separation between the CA neurons belonging to cytoarchitecturally different nuclei. In order to determine which CA neurons gave rise to striatal projections, the neostriatum was injected with True Blue (TB),and sections through the midbrain were processed for tyrosine hydroxylase (TH) and visualized by immunofluorescence. Neurons containing both TB and TH were observed in each of the CA cell groups mentioned above. The distribution of these cells confirmed organizational features that may be unique to the opossum's substantia nigra. In addition, different patterns of labeling resulted from caudate versus putamen injections, suggesting a rudimentary medial to lateral topography in the organization of nigrostriatal projections. Although our results suggest that the organization of midbrain CA neurons in the opossum is similar to that in placental mammals, it is clear that differences exist. Key words: tyrosine hydroxylase immunohistochemistry,double labeling,retrograde transport of

fluorescent dyes, substantia nigra, ventral tegmental area

It is generally recognized that the midbrain contains the largest number of catecholamine (CAI neurons in the mammalian brain and that most of them are located in the substantia nigra, the ventral tregmental area, the retrorubra1 area, the nucleus linearis, and the dorsal raphe nucleus. The degree of overlap among these cell groups has led to speculation that continuity exists in the distribution of CA neurons across the ventral midbrain (Dahlstrom and Fuxe, '64; Felten et al., '74; Crutcher and Humbertson, '78; Murray et al., '82; Pearson et al., '83). Numerous studies have revealed that CA neurons in each of the above areas project to the striatum in placental mammals (see Graybiel and Ragsdale, '79 and Carpenter, '84 for review) and that the projections from the substantia nigra and ventral tegmental area are topographically organized (Fallon and Moore, '78).

o 1991 WILEY-LISS, INC.

An earlier histofluorescence study documented the presence of monoaminergic cells in the midbrain of the North American opossum, a generalized marsupial (Crutcher and Humbertson, '78). In histofluorescence material, however, it is often difficult to distinguish catecholamine-like fluorescence from the fluorescence characteristic of serotonin. Because immunohistochemistry (Sternberger et al., '70) is more reliable for the identification of neurochemical markers than histofluorescence, we have reexamined the organization of CA neurons in the opossum's midbrain using an antibody against tyrosine hydroxylase (TH), the rate limitAccepted November 16,1990 Address reprint requests to James C. Hazlett, Dept. of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201.

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ing enzyme in the biosynthetic pathway for dopamine. It is likely that CA neurons revealed by TH immunohistochemistry are dopaminergic (Swanson and Hartman, '75; Swanson, '82). We also determined which of the CA neurons in the opossum's midbrain project to the striatum by combining the retrograde transport of the fluorescent marker True Blue (TB) with immunofluorescence for TH. To the best of our knowledge, this is the first such study in any marsupial and the organizational patterns described provide end points for developmental studies. Opossums are particularly useful for developmental studies because of their immaturity at birth, 12 days after conception, and their protracted development (Martin et al.,'78).

in PBS, mounted on gelatin-coated slides, and dried at 37°C overnight, dehydrated through graded alcohols, cleared in xylene, and coverslipped. The tissue sections were examined using a Leitz Orthoplan photomicroscope. Plots of the CA cells were made using a drawing tube attached to the microscope and representative sections were photographed. In order to reveal CA neurons giving rise to neostriatal projections, we used the retrograde transport of the fluorescent dye, True Blue (TB), in combination with a procedure for localizing tyrosine hydroxylase-like immunofluorescence in seven adult opossums. Each animal was anesthetized with sodium pentobarbital (32.5 m&g, IP), and in four cases multiple injections of 2%TB wlv in physiological saline were made into the striatum. In these injections, a total volume of 2.5-3.0 ~1 were delivered by pressure with a 5 - ~Hamilton 1 syringe fitted with a glass micropipet. In the MATERIALS AND METHODS remaining three animals, a single pressure injection of 0.06 Presumptive CA neurons were identified in five adult ~1 of 2% TB was made into the caudate nucleus using opossums by the indirect antibody peroxidase-antiperoxi- stereotaxic coordinates modified from Oswaldo-Cruz and dase (PAP) technique of Sternberger et al. ('70) utilizing Rocha-Miranda, '68. After a 10-day survival, each animal antibodies to tyrosine hydroxylase (TH) obtained from was anesthetized deeply and perfused transcardially with Eugene Technical. The animals were anesthetized deeply physiological saline (300 ml) and 4% paraformaldehyde in with intraperitoneal injections of sodium pentobarbital phosphate buffer (1,500 ml). The brains were removed and (120 mgikg) and perfused transcardially with saline (300 postfixed in the fixative containing 15%sucrose at 4°C for ml) followed by Zamboni's (Stephanini et al., '67) fixative 24 hours. (1,500 ml). Thirty-bm frozen sections were cut in the frontal plane The brains were immediately removed and placed in the and processed by the indirect immunofluorescence techsame fixative (4"C, 4-6 hours) and then placed in Soren- nique of Sawchenko and Swanson ('82). Briefly, the secson's phosphate buffer (pH 7.2) containing 15%sucrose at tions were incubated with the tyrosine hydroxylase anti4°C overnight. The midbrain and caudal diencephalon were body (diluted 1:4,000 in PBS with 0.3% TRI) for 48 hours at serially sectioned at 60 wm in the frontal plane with a 4°C (agitation for 1 hour) and then placed in fluoresceinfreezing microtome and the tissue sections were collected in isothiocyanate (FITC) conjugated goat antirabbit IgG antiborate-buffered saline (BBS, pH 8.2). The tissue sections body (diluted 1:30 and 1 : l O O in PBS and 0.3% TRI) for 1 were then incubated in the primary antibody directed hour at room temperature with agitation. Between each against TH (diluted 1:5,000 in BBS at pH 8.2, 0.5-0.75% step, the sections were thoroughly washed in PBS. Tissue bovine serum albumin [BSAI, 0.3% Triton X-100 [TRII) for sections were mounted and coverslipped with a PBS/ 2 days (4°C) with intermittent agitation totaling 2 hours. glycerine (1:3)solution. All sections were examined with a The tissue was then exposed to the sheep antirabbit IgG Leitz fluorescence microscope at excitation wavelengths of antiserum (diluted 1:300 in BBS-BSA-TRI, 1 hour, room 340-380 nanometer for TB and 450-490 nanometers for temperature, constant agitation) followed by the rabbit FITC. The locations of TB, FITC and TB + FITC labeled peroxidase-antiperoxidase complex (diluted 1:1,000 in BBS- neurons were plotted with an X-Y plotter interfaced to ii BSA, 1 hour, room temperature with constant agitation). fluorescence microscope stage. In addition, the location and The sections were thoroughly washed in BBS after each cytological features of representative single and double. incubation. The tissue was placed in 0.05% 3,3',5,5'- labeled neurons were recorded photographically. diaminobenzadine tetrahydrochloride and 0.006% hydroNomenclature. The distribution of CA neurons in the gen peroxide in phosphate-buffered saline (PBS), for 3-20 opossum midbrain was comparable to that reported for the minutes at room temperature. The tissue was then washed rat (Dahlstrom and Fuxe, '64; Hokfelt et al., '84). Although

Abbreviations Ac Amy be

ecV Cd CP dbc Am fx ic IC IF IP 11 or LL 111 Lr MG

nucleus accumbens amygdaloid complex brachium conjunctivum central gray, ventral caudate nucleus cerebral peduncle superior cerebellar peduncle, decussation medial longitudinal fascuculus fornix internal capsule inferior colliculus (central nucleus) interfascicular nucleus interpeduncular nucleus lateral lernniscus oculomotor nerve root linear nucleus medial geniculate nucleus

MM ml oc peg PN

PTr Pu RaD RN SNc SNl SNr TgP TgPP TgV Tro rIII

lateral mammillary nucleus medial lemniscus oculomotor nucleus parabrachial pigmentosus nucleus paranigral nucleus transverse peduncular tract nucleus putamen dorsal raphe nucleus red nucleus substantia nigra pars compacta substantia nigra pars lateralis substantia nigra pars reticulata deep tegmental area pedunculopontine tegmental area ventral tegmental area trochlear nucleus oculomotor nerve

CATECHOLAMINE PROJECTIONS TO OPOSSUM NEOSTRIATUM we have used the terminology of Oswaldo-Cruz and RochaMiranda ('68) for many areas of the opossum brain, we have provided an alternative terminology for the ventral tegmental area (based on studies by Taber, '61 and Berman, '68 in the cat and Phillipson, '79 in the rat) and the deep tegmental reticular formation (after Berman, '68).

RESULTS Distribution of tyrosine hydroxylase immunoreactive (TH-I) neurons in the midbrain TH-I neurons were located in midline cell groups, the deep tegmentum, the substantia nigra, and the ventral tegmental area, and the following description focuses on individual nuclei in these regions. Plots and photomicrographs documenting the locations of TH-I neurons are provided in Figures 1-5. Dorsal midline group. In the dorsal midline, TH-I neurons were located in two contiguous nuclei. The caudalmost group occupied the rostral one-third of the dorsal raphe nucleus (RaD in Figs. 1A,B, 2A,B), whereas the rostral group was located in the pars ventralis of the central gray matter (GCVin Figs. IC-F, 3A,B, 4A,B). In the RaD, most TH-I cells were located in ventromedial portions of its medial subdivision, and there was no obvious separation between these cells and the TH-I neurons of the subjacent nucleus linearis (Fig. 1B). The few TH-I cells in the lateral subdivision of the RaD were locatedjust dorsal and dorsolatera1 to the trochlear nucleus. The immunostained cell bodies in RaD were oval to fusiform with several, sparsely branched dendrites (Fig. 2B), many of which coursed either ventrally in the midline or dorsolaterally around the ventral aspect of the aqueduct. At this level, a few TH-I cells were also present just ventral to the cerebral aqueduct (Fig. lA,B). It was unclear whether these neurons were part of the dorsal raphe nucleus or a caudal extension of the TH-I group in the central gray matter. TH-I neurons in the ventral part of the central gray (GCv) first appeared as a recognizable cell group a t levels through the oculomotor nucleus (Figs. lC, 3A,B), although they may be present more caudally (Fig. lA,B). For the most part, these neurons formed a small, midline cluster immediately ventral to the cerebral aqueduct (Figs. 3A,B, 4A,B).This position was maintained throughout the rostral one-half of the midbrain and into the caudal diencephalon. The cell bodies of the TH-I neurons in the central gray were similar to those in the dorsal raphe nucleus with respect to shape and numbers of dendrites (compareFigs. 2B, 3B, 4B). Ventral midline group. We have identified two groups of TH-I neurons in the ventral midline. The dorsal cluster corresponds to the nucleus linearis (Oswaldo-Cruz and Rocha-Miranda, '68) and the ventral group to the nucleus interfascicularis. The former, because of its contiguity with the dorsal raphe nucleus, is included with the midline TH-I group. The interfascicular nucleus, in contrast, appears more closely associated with the ventral tegmental area (TgV). The dopaminergic neurons in both nuclei have been included in the current definition of the A10 dopamine region in the rat (see Phillipson, '79; Hokfelt et al., '84). Nucleus linearis (LR). The nucleus linearis (LR) extended from the trochlear nucleus, caudally, to caudal mammillary levels, rostrally (Fig. 1A-F) and TH-I neurons were distributed fairly evenly throughout its length. Caudally (Fig. 1A,B), TH-I neurons in the nucleus linearis

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overlapped similarly immunoreactive neurons in the overlying dorsal raphe nucleus, whereas more rostrally, they intermingled with those in the adjacent TgV and deep tegmental reticular formation (Fig. 1C,E). Most of the TH-I perikarya in the nucleus linearis were round to fusiform and had 2-4 primary dendrites (Figs. 2C,D), some of which coursed ventrally toward the interpeduncular nucleus. Ventrolateral nuclei. The TH-I neurons in the ventrolateral midbrain were distributed within the deep tegmental reticular formation, the TgV and the substantia nigra. Of these, the deep tegmental reticular formation was the largest in size and included the deep tegmental area (TgP) proper, the pedunculopontine tegmental area (TgPP), and an area bordering the ventral aspect of the red nucleus. Deep tegmental reticular formation. TH-I neurons appeared in moderate numbers in the TgP and TgPP at the level of the trochlear nucleus (Figs. lA, 2E). At caudal oculomotor levels (Fig. lB), TH-I neurons were scattered among the axons of the brachium conjunctivum and medial lemniscus (Fig. ZC), as well as between these bundles. TH-I cells along the ventral margins of this area marked the caudal extents of the substantia nigra (Fig. lB, small black star) and the TgV (see curved black arrow in Figs. lB, 2C). In general, this region appeared to be loosely organized with a moderate number of TH-I cells scattered within a diffuse plexus of TH-I axons and dendrites. The dendrites of these cells were long and sparsely branched. Farther rostrally (Fig. lC), it was difficult to distinguish TH-I neurons in the TgPP and TgP from those in the area bordering the ventral part of the red nucleus. Since the latter area, bounded by the red nucleus dorsally, the nucleus linearis medially and the TgV and substantia nigra ventrally, has not been named in the opossum, we have designated it as the ventral perirubral reticular formation. TH-I cells in this region were numerous caudally (Fig. lC), but decreased in number rostrally where they blended with immunostained neurons in the TgV and substantia nigra (particularly the dorsal aspect of the pars compacta and the more medial portions of the pars reticulata-Fig. lC,D). At oculomotor nerve levels (Fig. lD), the lateral portion of the ventral perirubral region was devoid of immunostained neurons, resulting in an absence of TH-I neurons between it and pars lateralis of the substantia nigra. Farther rostrally, TH-I neurons were present adjacent to the ventral half of the red nucleus (Fig. 1E). For the most part, the perirubral TH-I cell bodies were multipolar (Fig. 3C,F) and gave rise to 3-6 primary dendrites. Whereas some of the immunostained dendrites were severed near their cell bodies, suggesting that they coursed rostrally or caudally, numerous examples of lengthy dendritic arbors were observed. Substantia nigra (SN). All three subdivisions of the substantia nigra contained TH-I neurons. The pars compacts, as defined by Oswaldo-Cruz and Rocha-Miranda, '68, was first recognized with certainty at levels near the caudal portion of the red nucleus (Fig. 1C) where it contained a small but dense aggregate of TH-I cells clustered next to the ventromedial aspect of the cerebral peduncle. TH-I neurons of pars compacta maintained this position at progressively more rostral levels (Fig. lD,E) and exhibited areas of contiguity with the TH-I cells in pars reticulata, the overlying ventral perirubral reticular formation, and the medially adjacent TgV. The TH-I cells dorsal to the cerebral peduncle (Fig. lC,D) were located within either pars reticulata or pars lateralis accordingto the delineation of Oswaldo-

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Fig. 1. Plot of the distribution of TH-1 neurons on representative frontal sections through the middle and rostral mesencephalon (caudal A to rostral F) of an adult opossum. The small black star and curved black arrow in B indicate the caudal extents of the substantia nigra and ventral tegmental area, respectively. Each dot represents one TH-I cell. See abbreviations list.

CATECHOLAMINE PROJECTIONS TO OPOSSUM NEOSTRIATUM

Fig. 2. A. Photomicrograph of a frontal section a t the level of the trochlear nucleus from an opossum brain processed for TH immunoreactivity. The inset is enlarged in B. Bar = 0.5 mm; also applies to C. B. Higher magnification of inset in A illustrating TH-I neurons in the dorsal raphe nucleus. Bar = 50 pm; also applies to D, E, F. C. Photomicrograph of the ventrolateral quadrant of the section illustrated in A. Insets are shown at higher magnifications in D-F. The area enclosed by

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inset F represents the caudal portion of the ventral tegmental area. D. Higher magnification of inset C illustrating several TH-I neurons in the nucleus linearis. E. Higher magnification of inset in C illustrating several TH-I neurons in the dorsolateral aspect of the deep tegmental reticular formation (TgP). F. Higher magnification of inset in C illustrating TH-I neurons in the caudal portion of the ventral tegmental area (TgV).

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Fig. 3. A. Photomicrograph of a frontal section near the caudal end of the red nucleus from an opossum brain processed for TH. Inset is shown a t higher magnification in B. Bar = 0.5 mm; also applies to C. €3. Higher magnification of inset in A illustrating TH-I neurons in the midline portion of the ventral central gray area. Bar = 50 km; also applies to D-F. C. Photomicrograph of the ventrolateral quadrant of

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the section illustrated in A. Insets are shown at higher magnification in D-F. D. Higher magnification of inset in C illustrating TH-I neurons in pars lateralis of the substantia nigra. E. Higher magnification of inset in C illustrating TH-I neurons in pars reticulata ofthe substantia nigra. F. Higher magnification of inset in C illustrating TH-I neurons in the ventral perirubral reticular formation.

CATECHOLAMINE PROJECTIONS TO OPOSSUM NEOSTRIATUM Cruz and Rocha-Miranda ('68). At caudal mammillary levels (Fig. lF), TH-I cells of pars compacta were contiguous medially with and indistinguishable from those in the TgV. The TH-I cell bodies in the densely populated portions of pars compacta were round to oval and many of their discernable dendrites appeared to be transected near their origin (Fig. 4F). In contrast, TH-I neurons in areas adjacent to the perirubral reticular formation (Fig. 4E), pars reticulata of the substantia nigra and the TgV exhibited sparsely branched dendrites that could be traced for several hundred micrometers. Caudally, TH-I cells in pars reticulata appeared along the ventrolateral edge of the TgPP (Fig. 1B). This group expanded to form a loosely organized band of TH-I neurons along the tegmental aspect of the cerebral peduncle (Fig. 1C). At levels just caudal to the red nucleus, the distribution of TH-I cells in pars reticulata was continuous with that in pars compacta medially and the ventral perirubral reticular formation dorsally. In sections through the oculomotor nerve (Fig. lD), the only TH-I cells present in pars reticulata were located adjacent to pars compacta. The absence of TH-I neurons in the lateral part of pars reticulata created a discontinuity in the distribution of TH-I neurons between it and pars lateralis of the substantia nigra. This gap was in register with the one referred to previously in the overlying ventral perirubral reticular formation. Furthermore, there were few, if any, TH-I cells present among the fibers of the cerebral peduncle at any level. For the most part, the TH-I neurons of pars reticulata (Fig. 3E) were more widely separated from one another than those in pars compacta, and in many instances their dendrites could be followed from their cell bodies to their apparent terminations. Pars lateralis of the substantia nigra appeared at caudal rubral levels (Fig. 1C) and contained a small to moderate number of TH-I cells. It is important to note that this subdivision replaces pars reticulata at rostral midbrain levels (Fig. lE,F). The ventromedial shift of this subdivision was clearly evidenced by the change in position of the TH-I neurons from a position just dorsal to the dorsolateral edge of the cerebral peduncle at oculomotor nerve levels (Fig. 1D)to the tegmental aspect of the cerebral peduncle at medial geniculate (Fig. 1E) and mammillary levels (Fig. 1F). Like the TH-I neurons in pars reticulata, many of those in pars lateralis (Fig. 3D) gave rise to dendrites that could be traced for considerable distances in the transverse plane. Ventral tegmental area (TgV). The TgV contained TH-I neurons at all levels and could be divided into three cytoarchitecturallydistinct regions that corresponded closely to those reported in the rat (Phillipson, '79). Therefore, we utilize Phillipson's terminology to describe these subgroups: the nucleus parabrachialis pigmentosus, the nucleus paranigralis, and the nucleus interfascicularis. A small collection of TH-I cells, the caudal portion of the parabrachial pigmentosus nucleus, was first seen dorsomedial to the medial lemniscus (Fig. lB, small curved arrow). It remained in this position through oculomotor nerve levels (Fig. lD), being separated from the surface of the brainstem by the underlying paranigral nucleus. Farther rostrally, the paranigral nucleus disappeared and the pigmentosus subgroup expanded in a ventral direction (area indicated as PaPg in Fig. 1E). The latter group occupied this area throughout the rostral midbrain and caudal

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diencephalon (Fig. 1F). At several points, there were no obvious gaps between the distribution of TH-I cells in the pigmentosus nucleus and those within the overlyingventromedial perirubral area. Rostral to the middle part of the medial geniculate nucleus, the pigmentosus nucleus also became continuous with pars compacta of the substantia nigra. Throughout this region, most of the TH-I cell bodies were triangular or multipolar and gave rise to several sparsely but widely branched dendrites. This imparted a loosely organized appearance to the neuropil of this region (Figs. 2C,F, 4G, 5E). The two other TH-I subgroups were more limited in their distribution. The first, corresponding to the interfascicular nucleus, was located in the midline just dorsal to the interpeduncular nucleus (inset C in Fig. 4A, inset B in Fig. 5 ) . It contained a densely packed aggregate of moderately immunostained neurons. Just rostral to the interpeduncular complex, this cell group was located adjacent to the ventral surface of the brainstem (Fig. 1E). Farther rostrally, the number of TH-I neurons declined steadily before disappearing at levels through the rostral portion of the red nucleus. The second of the smaller subgroups of the TgV corresponded to the paranigral nucleus. This cell group first appeared at caudal rubral levels where it was located ventral and medial to the pigmentosus nucleus (Fig. 5A,C). It extended from the interfascicular nucleus dorsomedially to pars compacta ventrolaterally and contained several layers of oval or fusiform TH-I neurons (Fig. 5 0 . The long axis of many of these cells, together with many of their proximal dendrites, extended in dorsomedial-ventrolateral directions. Rostral to the oculomotor nerve, the paranigral nucleus decreased in size and became indistinguishable from the parabrachial pigmentosus nucleus. Mesencephalic origin o f TH projections to the striatum. In four animals, two with multiple TB injections that involved both the caudate and putamen and two with large, single injections centered in either the caudate nucleus or the putamen/amygdala, retrogradely labeled neurons (Fig. 6A-D, open circles and black stars) were observed in each of the previously identified TH-I cell groups. In general, the areas that contained the densest aggregates of TH-I neurons also exhibited the most retrogradely labeled cells. Figure 6 illustrates the results from one of the two animals that received multiple TB injections in which most of the head of the caudate nucleus, the dorsal aspect of the nucleus accumbens, the lateral edge of the septal complex, the caudal one-half of the putamen, and portions of the amygdaloid complex were included. Neurons containing both TB and FITC labeling (double-labeledneurons) were observed in the rostral third of the dorsal raphe nucleus and most of the nucleus linearis (Fig. 6A-D, 7A-D). All portions of the TgPP and the perirubral reticular formation contained double-labeled cells, and in caudal portions of this region, such cells showed marked differences in size and shape. The double-labeled cells near the lateral lemniscus were generally small and round to oval (Fig. 8A,B),whereas similarly labeled cells within the decussating bundles of the superior cerebellar peduncle were larger and fusiform or multipolar in shape (Fig. 8C,D). Farther rostrally, doublelabeled neurons in the ventral perirubral reticular formation were scattered widely at levels caudal and rostral to the oculomotor nerve (Figs. 6B,D, 8E,F, 9G,H). The TgV exhibited several variations in the distribution of double-labeled cells. The interfascicular subgroup (Fig.

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Figure 4

CATECHOLAMINE PROJECTIONS TO OPOSSUM NEOSTRIATUM 6C,D) contained multiple clusters (2-4 cells each) of small double-labeled neurons. Similar aggregates of such cells were detected in the paranigral group (Fig. 6B,C). Several of the labeled cell bodies were fusiform with a long axis oriented dorsomedially to ventrolaterally (Fig. 7E,F). Farther rostrally, these double-labeledcells blended with those in the parabrachial pigmentosus nucleus where they were few in number and widely separated (Fig. 9E,F). The substantia nigra also exhibited variations in the distributions of double-labeled cells. Pars compacta contained closely packed aggregates of double-labeled neurons, but in the other nigral subgroups such cells were more widely dispersed. The double-labeledcells in pars compacta occurred in multiple clusters (Figs. 6B-D, 9A,B), whereas similarly labeled cells in pars reticulata (Fig. 9C,D)and pars lateralis (Fig. 9I,J,K,L) were present either singly or in groups of 2-3 cells. A moderate number of double-labeled neurons in pars reticulata were present at levels through the caudal one-third of the red nucleus (Fig. 6B), although they were not observed more rostrally (Fig. 6C). The pars lateralis contained a small number of double-labeled cells (Fig. 6B-D). Each of the animals with small caudate injections exhibited several components of the larger labeling pattern. In the midline, double-labeled neurons were observed in the rostra1 portion of the dorsal raphe nucleus and in the underlying nucleus linearis. In addition, a few doublelabeled cells were present throughout the interfascicular nucleus, whereas the other two components of the TgV, the paranigral and the parabrachial pigmentosus nuclei, contained a moderate number of TB + FITC cells. A few double-labeledcells were present in the medial one-half of the deep tegmental reticular formation, but there was no apparent border between them and the ones located just dorsal to the densely packed portion of pars compacta. This latter area contained a moderate number of double-labeled cells throughout its extent. A few single-labeled TB cells were present in most areas containing double-labeledcells. This was most noticable in the TgV. FITC-labeledcells were present in numbers similar to those in the multiple injection cases. A medial to lateral topography in the distribution of retrograde labeling in the TgPP and substantia nigra was noted in cases with injections limited to the caudate nucleus or putamen-amygdala. The caudate injection labeled neurons in the pars compacta and the medial TgPP, whereas the putamen-amygdala injection labeled cells in pars compacts, pars reticulata, and pars lateralis of the substantia

Fig. 4. A. Photomicrograph of a frontal section of the midbrain at the level of the roots of the oculomotor nerve from an opossum brain processed for tyrosine hydroxylase. Insets are shown at higher magnifications in B and C. Bar = 0.5 mm; also applies to D. B. Higher magnification of inset in A illustrating TH-I neurons in the midline of the ventral central gray area. Bar = 50 pm; also applies to C, E-G. C. Higher magnification of inset in A illustrating TH-I neurons in the interfascicular nucleus of the ventral tegmental area. D. Photomicrograph of the left ventrolateral quadrant of the section illustrated in A. Note the virtual absence of TH-I structures in pars reticulata. Insets are enlarged in E-G. E. Higher magnification of inset in D illustrating TH-I neurons in the region between the perirubral area dorsally and pars compacta of the substantia nigra ventrally. F. Higher magnification of inset in D illustrating TH-I neurons in pars compacta of the substantia nigra. G. Higher magnification of inset in D illustrating TH-I neurons in the ventral tegmental area (parabrachial pigmentosus nucleus) just medial to the roots of the oculomotor nerve.

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nigra as well as within the lateral TgPP. Labeling in the dorsal and midline nuclei, the TgV and the ventral perirubral reticular formation appeared comparable in the two animals. The distribution of the FITC labeled neurons was identical to that observed in the immunoperoxidaseprepared material.

DISCUSSION In the opossum as in other mammals studied to date (rat-Dahlstrom and Fuxe, '64; Lindvall and Bjorklund, '74; Palkovits and Jacobowitz, '74; Hokfelt et al., '76, '84; Phillipson, '79; Swanson, '82; Descarries et al., '86; rabbitBlessinget al., '78; cat-Wiklund et al., '81; Maichon et al., '83; tree shrew-Murray et al., '82; monkey-Tanaka et al., '82; Garver and Sladek, '75; humans-Gasper et al., '83; Pearson et al., '83), midline and ventral nuclei of the midbrain contain CA neurons (see Graybiel and Ragsdale, '79, '83; Carpenter, '84 for review). Although the distribution of CA neurons in the midbrain appears comparable in different mammals, there are features that are particularly prominent or perhaps unique to the opossum. It is obvious in the opossum that regional variations exist in the distribution of loosely organized versus densely packed aggregates of CA cells. Several of these variations, especially noticeable among the ventral CA cell groups, indicate that areas of contiguity exist among the CA cells of different nuclei. We have also observed unusual features in the substantia nigra. These include: (1)gaps in the distribution of CA neurons, (2) a pars compacta that does not form a dorsal cellular lamina, and (3) nigral subdivisions that are arranged in a medial to lateral sequence.

Continuities in neuronal organization CA neurons of the ventrolateral cell groups can be subdivided according to density. They are densely aggregated within pars compacta as well as within the interfascicular and paranigral subdivisions of the TgV, but they are more widespread and loosely organized in other nuclei of the ventrolateral complex. It is obvious that differences in the density of CA cells do not honor the limits of specific nuclei. For instance, the retrorubral area contains only loosely organized CA neurons, whereas the TgV and the substantia nigra contain both loosely organized and densely packed cells. There is little separation between the CA cells in several nuclei of the ventral midbrain and loosely organized CA cells characterize the areas of contiguity. Examples of contiguity include that between the CA cells of the retrorubral area and those within the dorsal pars reticulata and dorsal pars compacta, as well as that between CA cells of the retrorubral area and the parabrachial pigmentosus nucleus. Contiguity between CA cells of the pigmentosus nucleus and the medial aspect of pars compacta was also present, although it is only obvious at midmedial geniculate levels where the pars compacta begins to lose its densely packed appearance. In contrast, CA cells in the interfascicular and paranigral nuclei, as well as within the densely packed portion of pars compacta, do not abut one another. The issue of contiguity has been discussed in a number of studies, each documenting approximations between CA cells of pars compacta and those in the TgV andfor the retrorubral nucleus (opossum-Crutcher and Humbertson, '78; rat-Dahlstrom and Fuxe, '64; Fallon and Moore,

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Fig. 5. A. Photomicrograph of the ventral mesencephalon including the ventral tegmental area at a level just caudal to the roots of the oculornotor nerve. The section was processed for TH. Insets are illustrated at higher magnifications in B-E. Bar = 0.5 mm. B. Higher magnification of inset in A showing TH-I neurons in the interfascicular nucleus (IF).Bar = 50 pm; also applies to C-E. C. Higher magnification of inset in A showing TH-I neurons in a portion of the paranigral

nucleus (PN). Cells indicated by arrowheads are oriented in the dorsomedial to ventrolateral directions. D. Higher magnification of inset in A showing TH-I cells in the nucleus linearis (Lr)just dorsal to the interfascicular nucleus. E. Higher magnification of inset in A showing TH-I neurons in the parabrachial pigmentosus nucleus (PaPg). Note the diffuse distribution of these cells compared to the TH-I neurons in the interfascicular nucleus.

'78; rabbit-Blessing et al., '78; cat-Poitras and Parent, '78; tree shrew-Murray et d.,'82; monkey-Felten et al., '74; Felten and Sladek, '83; humans-Pearson et al., '83). The proximity of CA neurons within the retrorubral area (considered coextensive with A8-Anden et al., '64, Dahlstroin and Fuxe, '64) and those within pars compacta has prompted suggestions that the retrorubral area is the

caudal continuation of pars compacta (Hubbard and DiCarlo, '73; Fallon and Moore, '78; Murray et al., '82). In the rat this continuity involves the dorsal tier (Gerfen, '86) or dorsal sheet (Fallon and Loughlin, '85) of pars compacta. This latter group exhibits several anatomical and neurochemical characteristics which set it apart from the remainder of pars compacta (Gerfen, '86).

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SNc

Fig, 6. Plot of the distribution of the various cell types labeled by injections of True Blue (TB) into the neostriatum followed by TH immunohistochemistry. The primary focus of the injection sites (solid black) and the diffusion area (shaded) are plotted in the drawings at the

upper right. The locations of single-labeled neurons containing TB or TH immunoreactivity (FITC),as well as double-labeled cells containing both TB and FITC, are plotted on representative coronal midbrain sections from caudal (A)to rostral (D).

Contiguity involving loosely organized cells was also apparent between CA cells in the midline and those in adjacent areas. They occur at the border between the dorsal raphe and linear nuclei (similar to that seen by Descarries et al., '86 in the rat) and, at several points, between the linear nucleus and the ventrolateral cell groups. In the latter situation, it is not clear that others have observed similar contiguity. Only a few studies have reported or illustrated close approximation between the midline and the ventrolateral dopamine cell groups (Swanson, '82; Maichon et al., '841,although Paxinos and Butcher ('85)

mentioned that the retrorubral fields, as depicted in the Paxinos and Watson ('82) atlas of the rat brain, should be extended to the midline caudal linear nucleus.

Discontinuities (gaps) in the distribution of CA neurons A gap in the distribution of CA neurons in the opossum midbrain occurs at the level of and slightly rostral to the roots of the oculomotor nerve (see Fig. 1D). The areas involved include pars reticulata of the substantia nigra and

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Fig. 7. Fluorescent photomicrographs of neurons (arrows) labeled by TB (illuminated at 340-380 nm) in the dorsal raphe nucleus (A), the nucleus linearis (C) and the paranigral nucleus (E)following an injection of TB into the caudate nucleus. The corresponding sections B,

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D, and F were photographed at 450-490 nm to visualize TH-I (FITC). Arrows point to FITC cells that also exhibited TB. Bar in A indicates 25 +m; applies to A-F. The directional markers indicate dorsal (Dor) and lateral (Lat) and apply to E and F.

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Fig. 8. Fluorescent photomicrographs of neurons (arrows) labeled by TB (illuminated at 340-380 nm) in the dorsolateral deep tegmentum (A), the deep tegmentum within the decussation of the superior cerebellar peduncle ( C ) and the ventral perirubral reticular formation

(E)following an injection of TB in the caudate nucleus. The corresponding photomicrographs B,D and F illustrate FITC neurons illuminated at 450490 n m to reveal TH immunoreactivity. Arrows point to FITC cells that also exhibited TB. Bar in A = 25 pm; applies to A-F.

the ventral perirubral reticular formation (rostral extension of A8). This gap appears particularly obvious because there is no dorsal pars cornpacta. To the best of our knowledge, such a pronounced gap has not been described in other species, although a similar discontinuity has been noted in the rat (Dahlstrom and Fuxe, '64; Hokfelt et al.,

'84) and the cat (Poitras and Parent, '78). In these species, most pars reticulata dopaminergic neurons are found at more caudal levels, and such cells gradually disappear from the rostral extension of A8 at oculomotor levels (Dahlstrom and Fuxe, '64).In both species,the more rostral portions of pars reticulata contain only a few widely scattered CA

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Figure 9

CATECHOLAMINE PROJECTIONS TO OPOSSUM NEOSTRIATUM neurons and the ventrally directed dendrites of pars compacts cells. In the opossum, this interval is devoid of CA neurons, although it is filled by nigrotectal and nigrothalamic neurons many of which appear to be GABAergic (Hazlett et al., '87a).

Organization of the substantia nigra As in the other nonprimates, the opossum substantia nigra is composed of three subdivisions, each providing CA projections to the striatum. However, the organization of these subdivisions exhibits features that may be unique to the opossum. The most striking include the dense packing of CA neurons medial to the cerebral peduncle and their sparsity dorsal to it. It appears, therefore, that the opossum nigra is oriented with pars compacta located ventromedially, pars reticulata in an intermediate position, and pars lateralis positioned dorsolaterally. Whereas this pattern is different from the dorsal to ventral organization common to eutherian mammals, it agrees with that described for the opossum by Oswaldo-Cruz and Rocha-Miranda ('68). The above organization is confirmed by the connections of these subdivisions (Hazlett et al., '87a). Briefly, our results revealed overlap between nigrostriatal neurons in the caudal half of pars reticulata (as defined by OswaldoCruz and Rocha-Miranda, '68) and a large number of nigrotectal neurons. Furthermore, the largest accumulation of nigrostriatal cells was located ventromedial to the peduncle. We conclude, therefore, that pars compacta does not form a separate cellular lamina overlying pars reticulata in the opossum. Instead, it is located primarily medial to the cerebral peduncle. For the present, we consider the TH-I, striatally projecting neurons that are intermixed with the nigrotectal cells as part of pars reticulata.

Connections of the midbrain catecholamine-containing neurons The studies described herein are the first to report a neurochemically defined mesostriatal projection in any marsupial and suggest that CA projections from the midbrain to the striatum of the opossum are similar to those reported in eutherian mammals, particularly the rat (see below for references). As in other species, these projections

Fig. 9. A. Fluorescence photomicrograph of neurons in pars compacts of the substantia nigra retrogradely labeled by injections of true blue (TB) into the ipsilateral caudate nucleus (illuminated at 340-380 nm). Bar = 25 pm; also applies to B-L. B. Photomicrograph of the same neurond field (arrows) shown in A, but illuminated at 450-490 nm to reveal TH immunoreactivity. C. Photomicrograph of neurons in pars reticulata labeled by injections of TB into the ipsilateral putamen (illuminated at 340-380 nm). D. Photomicrograph of the same neurons (arrows) shown in C, but illuminated at 450-490 nm to reveal TH immunoreactivity. E. Photomicrograph of a neuron in the parabrachial pigmentosus nucleus of the ventral tegmental area labeled by the TB injection described in A (illuminated at 340-380 nm). F. Photomicrograph of the same cell shown in E, but illuminated at 450-490 nm to reveal TH immunoreactivity. G . Photomicrograph of a neuron in the ventral perirubral reticular formation just dorsal to pars lateralis labeled by the TB injection described in C (illuminated at 340-380 nm). H. Photomicrograph of the same cell shown in G, but illuminated at 450-490 nm to reveal TH immunoreactivity. I and K. Photomicrographs of neurons in pars lateralis labeled by the TB injection described in C (illuminated at 340-380 nm). J and L. Photomicrographs of the same cells (arrows) shown in I and K, but illuminated at 450-490 nm to reveal TH immunoreactivity.

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are organized topographically. It is of interest that non-CA neurons of the midbrain may also innervate the striatum: the chemical signature of such neurons is not known, although some of them may be GABAergic (Deniau et al., '78; Gerfen et al., '87) or peptidergic (Hokfelt et al., '80; Fallon et al., '83). Previous studies indicate that each of the midbrain CA cell groups gives rise to projections that terminate in one or more areas of the forebrain (Anden et al., '64; Dahlstrom and Fuxe, '64; Bedard et al., '69; Moore et al., '71; Ungerstedt, '71; Lindvall and Bjorklund, '74; Nauta and Domesick, '76; Fallon and Moore, '78; Van der Kooy and Wise, '80; Fallon and Loughlin, '82; Swanson, '82; Trulson et al., '85; Descarries et al., '86; see Graybiel and Ragsdale, '79, '83; Carpenter, '84 for review), but the most pertinent to the present study are the projections to the neostriatum. With regard to the midline nuclei, Descarries et al. ('86) have demonstrated dopaminergic projections from the dorsal raphe nucleus and the nucleus linearis to the striatum in the rat, and it is clear from our study that comparable projections exist in the opossum. Bjorklund and Lindvall ('84) suggest that dopaminergic cells in the rostra1 part of the periaqueductal gray project to ventral rather than dorsal striatal structures. Since CA neurons were labeled in that area after injections that included part of the ventral striatum (nucleus accumbens, lateral septal nucleus, amygdaloid complex or bed nucleus of the stria terminalis) but were not labeled after injections limited to the dorsal striatum, we assume that comparable projections exist in the opossum. The ventrolateral cell groups are well-known sources of dopaminergic projections to both striatal and limbic targets (see Graybiel and Ragsdale, '79, '83; Carpenter, '84 for review). Striatal projections from the retrorubral area have been reported in the mouse (Mattiace et al., '891, the rat (Nauta et al., '78; Swanson, '821, and the cat (Royce, '78; Szabo, '80a), and Swanson ('82) indicated that approximately two-thirds of the retrorubral neurons projecting to the nucleus accumbens are dopaminergic. Numerous studies have focused on the organization of striatal projections from the substantia nigra and the TgV (the opossum-Hazlett et al., '84; Hazlett and Martin, '86; the mouse-Mattiace et al., '89; the rat-Nauta et al., '74, '78; Robertson and Travers, '75; Faull and Mehler, '78; Fallon and Moore, '78; Nauta and Domesick, '79; Van der Kooy and Wise, '80; Veening et al., '80; Swanson, '82; the cat-Royce, '78; Vandermaelen et al., '78; Szabo, '80a; the monkey-Szabo, '80b). There is general agreement that pars compacta and adjacent portions of the TgV are the largest contributors to mesostriatal projections, although in nonprimate mammals, pars reticulata and pars lateralis are also involved. Colocalization studies (Van der Kooy and Wise, '80; Swanson, '82) have demonstrated dopaminergic projections from the TgV, pars compacta, and portions of pars reticulata to the dorsal striatum, the nucleus accumbens, the amygdala, and the lateral septal nucleus. The present study documents the presence of striatal projections from dopaminergic neurons within pars compacta of Oswaldo-Cruz and Rocha-Miranda ('681, as well as within the caudal part of pars reticulata and throughout the pars lateralis. Projections from the TgV to the striatum have been described in the mouse (Mattiace et al., '89) and the rat (Fallon and Moore, '78; Faull and Mehler, '78; Van der Kooy and Wise, '80; Swanson, '82) and their dopaminergic

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character confirmed (Van der Kooy and Wise, '80), but the TgV also contributes dopaminergic projections to the nucleus accumbens, the septa1 nuclei, and the amygdaloid complex (Fallon and Moore, '78; Swanson, '82). Although we cannot confirm the latter connections in the opossum, it is clear that dopaminergic neurons in the TgV innervate the caudate nucleus. Many investigators (Faull and Mehler, '78; Fallon and Moore, '78; Swanson, '82; Carpenter, '84) have reported that mesostriatal projections are organized topographically. We have confirmed a rudimentary medial to lateral topography in nigrostriatal and retrorubrostriatal projections of the opossum similar to that reported in the rat by Fallon and Moore, '78. In addition, it appears that the nigroputamen projection emanates largely from the more loosely organized cells, whereas most of the caudate projection arises from densely packed neurons. In confirming the medial to lateral organization of nigrostriatal projections, we have shown that the caudate nucleus receives its nigral projections primarily from pars compacta with additional contributions from the dorsal raphe and linearis nuclei, the medial aspect of the retrorubra1 area, and the TgV. We have assumed that the striatal projections from pars reticulata, pars lateralis, and the lateral portion of the retrorubral area terminate, at least in part, in the putamen. Such connections were implied from cases with large putameniamygdala injections. As a partial confirmation of these connections, we report the results of several small horseradish peroxidase injections limited to the dorsal putamen (unpublished observations). In each case, retrogradely labeled cells were present in pars reticulata, pars lateralis, the lateral retrorubral area, the TgV (all subdivisions) as well as within the dorsal raphe and linearis nuclei. In addition, there were a few labeled cells in the more dorsal aspect of pars compacta. Whether such neurons are CA-immunoreactive was not determined, although their location and configuration suggests that they contribute to the CA projection to the putamen.

Conclusions Our results suggest that the organization of CA neurons in the midbrain of the opossum is similar to that described for rats, cats, and monkeys. Several differences were noted, however, and future studies will be required to determine whether such dissimilarities are unique to marsupials. The opossum is born in a very immature state, 12-13 days after conception, and its nervous system undergoes a protracted development, whereas the newborn is maintained by the mother in an external pouch. Preliminary evidence (Martin et al., '89) suggests that the development of the major projections to the striatum, including the nigrostriatal projection, occurs postnatally. We hope to take advantage of the opossum's embryology to study the development of the systems described herein and define their role in the ontogeny of motor function.

ACKNOWLEDGMENTS

I

The authors thank Mrs. Mary Ann Jarrell for technical assistance and help with typing the manuscript. We also thank Drs. Jerald Mitchell and Jose Rafols for helpful comments. This study was supported by a grant from the Michigan Eye Bank and Transplantation Center to J.C.H. and USPHS Grant NS-25095 to G.F.M.

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