Dorsal striatopallidal system in anurans

THE JOURNAL OF COMPARATIVE NEUROLOGY 468:299 –310 (2004)

Dorsal Striatopallidal System in Anurans HEIKE ENDEPOLS,1* KATJA RODEN,1 HARALD LUKSCH,1 URSULA DICKE,2 1 AND WOLFGANG WALKOWIAK 1 Institute of Zoology, University of Cologne, D-50923 Ko¨ln, Germany 2 Brain Research Institute, University of Bremen, D-28334 Bremen, Germany

ABSTRACT The dorsal striatopallidal system of tetrapods consists of the dorsal striatum (caudateputamen in mammals) and the dorsal pallidum. Although the existence of striatal and pallidal structures has been well documented in anuran amphibians, the exact boundaries of these structures have so far been a matter of debate. To delineate precisely the dorsal striatopallidal system of anurans, we used quantitative analysis of leucine-enkephalin immunohistochemistry (in Bombina orientalis, Discoglossus pictus, Xenopus laevis, and Hyla versicolor), retrograde neurobiotin tracing studies (injections in the central and ventromedial thalamic nuclei in H. versicolor), and double-labeling tracing studies (injections in the lateral forebrain bundle and the caudal striatum in B. orientalis). Immunohistochemistry revealed that enkephalin-positive neurons are located mainly in the rostral and intermediate striatum. Neurobiotin tracing studies demonstrated that neurons projecting to the central and ventromedial thalamic nuclei are found in the intermediate and caudal striatum. Doublelabeling studies revealed that the population of neurons in the rostral and intermediate striatum innervating the caudal striatum is separated from neurons projecting into the lateral forebrain bundle. Neurons that project to both the caudal striatum and the lateral forebrain bundle are found only in the dorsal part of the intermediate striatum. Taken together, our results suggest that the rostral striatum of anurans is homologous to the striatum proper of mammals, whereas the caudal striatum is comparable to the dorsal pallidum. The intermediate striatum represents a transition area between the two structures. J. Comp. Neurol. 468:299 –310, 2004. © 2003 Wiley-Liss, Inc. Indexing terms: amphibians; striatum; pallidum; immunohistochemistry; neurobiotin tracing; quantitative evaluation

Because animal models have become more and more attractive for the study of Morbus Parkinson and other human diseases related to the basal ganglia, it is important to find structural as well as functional homologies between the basal ganglia of mammals and those of the other tetrapod groups, viz. birds, reptiles, and amphibians. In anuran amphibians, behavioral studies have shown that lesions of the striatum change or impair visually and acoustically guided behavior (Matsumoto et al., 1991; Walkowiak et al., 1999), suggesting that the amphibian basal ganglia may be crucial parts of the motor system. It now seems evident that the anatomical organization of the basal ganglia is also fundamentally similar among vertebrates (Reiner et al., 1984, 1998; Parent, 1986; Russchen et al., 1987a,b; Medina and Reiner, 1995; Marı´n et al., 1998a,c; Smeets et al., 2000). For anurans, some studies have homologized subpallial telencephalic structures with the dorsal and ventral striatopallidal systems defined in mammals. In the classical view (Northcutt and Kicliter, 1980; see Fig. 1A), based on © 2003 WILEY-LISS, INC.

Nissl-stained sections of Rana catesbeiana, the striatum was subdivided into a dorsal and a ventral part. It covered the ventrolateral telencephalic wall, whereas the nucleus accumbens was situated at the ventral tip of the telencephalic ventricle. Rostrally, striatum and nucleus accumbens were demarcated by the olfactory bulb; caudally, the dorsal striatum was replaced by the lateral amygdala. The

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Wa 446/4. Harald Luksch’s current address is Institute of Biology II, RWTH Aachen, Kopernikusstrasse 16, D-52074 Aachen, Germany. *Correspondence to: Heike Endepols, Institute of Zoology, University of Cologne, Weyertal 119, D-50923 Ko¨ln, Germany. E-mail: [email protected] Received 9 December 2002; Revised 24 July 2003; Accepted 25 September 2003 DOI 10.1002/cne.11006 Published online the week of November 17, 2003 in Wiley InterScience (www.interscience.wiley.com).

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ventral striatum persisted ventrolaterally of the lateral amygdala and stretched as far caudally as to the rostral boundary of the anterior entopeduncular nucleus. Caudally, the nucleus accumbens bordered the medial amygdala. A dorsal and ventral pallidum had not been described. This classical view has been altered after a recent series of hodological and immunohistochemical studies (Marı´n et al., 1997a,b, 1998b,c). According to this view, the nucleus accumbens is located in the ventromedial telencephalic wall along the rostral one-third of the telencephalic hemisphere. The ventral pallidum occupies the most ventromedial aspect of the telencephalon, covering the caudal twothirds of the hemisphere. With regard to the exact borders of striatum and dorsal pallidum, however, there have been different views. By using immunohistochemical staining of substance P, enkephalin, and dopamine/tyrosine hydroxylase as well as hodological data, the pallidum was defined at caudal and superficial levels of the striatum, similar to the location of the pallidum in cartilaginous fish and lungfish (Medina and Reiner, 1995; Reiner et al., 1998; see Fig. 1B). By using exactly the same methods as the former authors, Marı´n et al. (1998b,c; Fig. 1C) placed the dorsal pallidum in the ventromedial part of the caudoventral striatum, adding the anterior entopeduncular nucleus and ventrolateral parts of the medial amygdala to this structure as well. Recently, the expression of genes acting in early regional specification (e.g., x-Nkx2.1) or in cell determination (e.g., x-Lhx7) has been used to delineate pallial and subpallial telencephalic regions in Xenopus. It could be demonstrated that primordia of the striatum (lateral ganglionic eminence) and the pallidum (medial ganglionic eminence) are present in the developing anuran brain (Bachy et al., 2001, 2002; Brox et al., 2002; Gonza´lez et al., 2002). The anterior entopeduncular area appears, based on gene expression, as a caudal continuation of the pallidal region (Brox et al., 2002; Gonza´lez et al., 2002). However, the border between striatum and dorsal pallidum could not be delineated with the help of gene expression patterns, because neurons migrate from the medial into the lateral ganglionic eminence, forming striatal interneurons (Gonza´lez et al., 2002). Thus, additional studies are necessary to clarify the rostral and caudal margins of the anuran dorsal pallidum. Here we apply two methods to distinguish between striatum and dorsal pallidum in anurans. The first is a quantitative evaluation of the distribution of neurons

Fig. 1. Comparison of telencephalic nomenclature proposed by different authors. Depicted are the left halves of transverse sections (in C, only subpallial structures are shown); they are drawn after the original figures. Every row represents roughly the same rostrocaudal level, which is indicated in the insets. The first column (A) shows the classical nomenclature put forward by Northcutt and Kicliter (1980; Rana catesbeiana). In the other columns, brain nuclei are indicated by boldace and underscoring when their boundaries or nomenclature differ from the classical scheme (B: no species indicated; C: Rana perezi; D: Hyla versicolor). For abbreviations see list. Scale bars ⫽ 500 ␮m.

immunoreactive for enkephalin, because, in mammals, enkephalinergic neurons are found in the striatum but not in the dorsal pallidum (Graybiel, 1990b). Second, a

Abbreviations A Aa Acc Al Am BN BST C CO DB DP Ea Ep La LFB Pd

anterior thalamic nucleus anterior amygdala nucleus accumbens lateral amygdala medial amygdala bed nucleus of the pallial commissure bed nucleus of the stria terminalis central thalamic nucleus chiasma opticum diagonal band of Broca dorsal pallidum anterior entopeduncular nucleus posterior entopeduncular nucleus anteriolateral thalamic nucleus lateral forebrain bundle dorsal pallium

Pl Pld Plv Pm PoA SC Sl Sm Std Str Stv Ven VH VL VM VP

lateral pallium dorsal part of the lateral pallium ventral part of the lateral pallium medial pallium preoptic area suprachiasmatic nucleus lateral septum medial septum dorsal part of the striatum striatum ventral part of the striatum ventricle ventral hypothalamic nucleus ventrolateral thalamic nucleus ventromedial thalamic nucleus ventral pallidum

ANURAN DORSAL STRIATOPALLIDAL SYSTEM quantitative analysis of retrogradely labeled neurons after tracer injections into thalamic nuclei was performed, because the thalamus of mammals is innervated by the dorsal pallidum but not by the striatum (Parent, 1990; Smith and Bolam, 1990; Nakano et al., 2000). Additionally, double labeling of striatal neurons was carried out to contribute to the knowledge of the intrinsic organization of the anuran basal ganglia.

MATERIALS AND METHODS For leucine-enkephalin immunohistochemistry, we used five Xenopus laevis, five Bombina orientalis, five Discoglossus pictus and five Hyla versicolor. The antibody was polyclonal (rabbit) and was obtained from IBL (Hamburg, Germany). The animals were lethally anesthetized in a 0.2% solution of tricaine methanesulfonate (MS 222, Sigma-Aldrich, St. Louis, MO; Ohr, 1976) in tap water and subsequently perfused transcardially with cold 0.1 M sodium phosphate buffer (PB), pH 7.4, followed by a solution of 4% paraformaldehyde in PB. Brains were removed, postfixed for 4 –7 hours, and rinsed in PB overnight. After an immersion in 15% sucrose in PB for 1 hour and subsequent immersion in embedding medium (Reichert-Jung) for 30 minutes, brains were frozen rapidly. Transverse or sagittal sections (15–25 ␮m thick) were cut on a cryostat (Reichert-Jung Frigocut 2800) and mounted onto chromalum-gelatinized slides; every fifth section was collected on a separate slide for specificity testing. The sections were air dried at room temperature for 1 hour and rinsed three times (10 minutes each) in PB, followed by an immersion in 0.5% Triton X-100 (Serva) in PB for 30 minutes. After several rinses in PB, sections were incubated with normal serum (1:20 in PB) for 30 minutes on a shaker and subsequently incubated with the primary antibody [both 1:2,000 in PB containing 1% bovine serum albumin (BSA)] at 4°C overnight or for 2 hours at room temperature; the control sections were incubated with PB containing 1% BSA. After three rinses in PB (10 minutes each), the binding sites of the primary antibody were visualized with the PAP technique (secondary antibody 1:100, 30 minutes; PAP complex 1:100, 30 minutes). The chromogen for the reaction was diaminobenzidine (DAB; Boeringer Mannheim, Mannheim, Germany), with a heavy-metal intensification following the protocol of Adams (1981); the H2O2 was provided by a glucose-oxidase reaction (Shu et al., 1988). After the DAB procedure, sections were dehydrated in ethanol and coverslipped with Corbit (Hecht, Kiel-Hassee, Germany). For neurobiotin tracing studies, we used the isolated brain preparation of eight Hyla versicolor. Details of brain dissection are described elsewhere (Luksch et al., 1996). Briefly, animals were deeply anesthetized in 0.2% MS 222 (Sigma-Aldrich; Ohr, 1976), cooled to a body temperature of 5°C, and perfused transcardially with 40 ml ice-cold oxygenated Ringer’s solution (Straka and Dieringer, 1993). The brain was removed from the skull prior to tracer injection by a ventral approach, and then placed into an electrophysiological recording chamber perfused with Ringer’s solution. Ten percent biotin ethylendiamine (Neurobiotin; Molecular Probes, Eugene, OR) in 0.3 sodium acetate solution was used to fill a glass micropipette (2–3 M⍀) and injected with positive current (150 –200 nA) for 30 minutes. Injection sites were the central and ventromedial thalamic nuclei. After tracer application, the brain was kept for 48 hours at 7°C to allow transport and

301 then fixed with 4% paraformaldehyde plus 1.25% glutaraldehyde in PB overnight. After being washed in PB for at least 24 hours, brains were embedded in 4% agar (Merck) in PB and cut into 50-␮m transverse sections with a vibratome (D.S.K. Microslicer DTK-3000). Sections were directly mounted onto chromalum-gelatinized slides, dried at 37°C, rinsed in PB for 10 minutes, and incubated with 2% streptavidin-horseradish peroxidase (HRP; Amersham, Arlington Heights, IL) ⫹ 0.5% Triton X-100 (Serva, Heidelburg, Germany) in PB overnight. After several washes in PB, HRP was visualized with the DAB procedure (see above). After staining, sections were dehydrated in ethanol and coverslipped with Corbit (Hecht). For the description of labeled structures, we followed the nomenclature of Northcutt and Kicliter (1980). Double-labeling studies were performed on the isolated brain preparation (see above) of four Bombina orientalis. After isolation of the brain, tracers were applied as crystals on the tip of sharp glass micropipettes, where the tracer had been recrystallized from a saturated solution in distilled water. We used 10-kD Oregon green 488conjugated dextran amine (OGDA; Molecular Probes) for tracer applications into the lateral forebrain bundle at the rostrocaudal level of the anterior thalamic nucleus and 10-kD tetramethyl rhodamine-conjugated dextran amine (TMRDA; Molecular Probes) for applications in the caudal striatum. The brain was kept in Ringer’s solution at 7°C for 48 hours and was then fixed overnight in a solution of 4% paraformaldehyde and 1.25% glutaraldehyde in PB. After several washes in PB, the brain was immersed in 7.5% sucrose in PB, 15% sucrose in PB, 15% sucrose ⫹ embedding medium (1:1), and embedding medium (Reichert-Jung) for 1 hour each. Then, the brain was frozen in fresh embedding medium and cut into 25-␮m transverse sections on a cryostat (Reichert-Jung Frigocut 2800). To block autofluorescence of glutaraldehyde, sections were immersed in 0.25% sodium borohydride (Applichem) in PB for 15 minutes and then coverslipped in glycerol gelatin (7 g gelatin in 42 ml water ⫹ 50 g glycerol ⫹ 0.5 g phenol). Sections of rostral and intermediate striatum were examined with a laser scan microscope (Zeiss LSM 510) in confocal mode. To visualize labeled neurons, every section was divided into 10 focal planes, which afterward were reconstructed. OGDA-, TMRDA-, and double-labeled neurons were counted and allocated to the dorsal or ventral striatum. For quantitative analyses, the striatum was divided into rostral, intermediate, and caudal parts. In the case of transverse sections (all tracing studies and some immunostainings), this was done by dividing the telencephalic sections containing the striatum into three equal parts. When many neurons were labeled, each part was further divided, which resulted in six or nine striatal divisions altogether. In the case of sagittal sections (most of the immunostainings), rostral, intermediate, and caudal parts of the striatum were demarcated with the help of a micrometer eyepiece. Immunostained or retrogradely labeled cells were counted on each section by using a ⫻20 objective. Whereas neurons in DAB-stained sections were counted directly from the microscopic image, fluorescent neurons (double-labeling tracing studies) were counted from confocal images. In enkephalin immunohistochemistry and retrograde tracing from thalamic nuclei, relatively few neurons were labeled in the striatum. It was therefore possible to analyze serial sections and to evaluate whether a labeled cell body was visible on two adjacent sections. To

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rule out that a neuron was counted twice, we registered only those neurons that were not visible in the following section (physical disector; for review see von Bartheld, 2002). In the double-labeling tracing studies, many cell bodies were stained in the striatum, and analysis of serial sections was not possible. Stereological methods may not be necessary to determine a rough percentage of double labeled cells (Saper, 1996), but we decided to use a simple correction formula, the Abercrombie-Floderus method (assuming that all particles are round and of the same size) to calculate a correction factor: C ⫽ T/(T – 2R ⫹ H) (Floderus, 1944; Abercrombie, 1946; Clarke, 1993), where T ⫽ section thickness (obtained by focusing), 14 –22 ␮m; R ⫽ height of “lost caps” (minimum distances that the upper and lower limits of the cell body have to protrude into the section in order to be detected), 0.5 ␮m; and H ⫽ height of cell bodies, 10 ␮m. The number of neurons counted in every section was multiplied by the correction factor C, which ranged from 0.61 to 0.67. Photomicrographs were recorded with two digital cameras (Pixera PVC 100C and Nikon Coolpix 4500) mounted on a Leica DMLB microscope. Brightness and contrast of all images (photomicrographs and laser scan images) were adjusted using Corel Photopaint 11. All experiments complied with the principles of animal care (publication No. 86-23, revised 1985) of the National Institute of Health and also with the current laws of the country in which the experiments were performed.

RESULTS Leu-enkephalin immunohistochemistry Although the quality of immunostaining was comparable in all brains as assessed by labeled structures in other brain areas, the number of immunoreactive cells in the striatum varied greatly among individuals. Total cell counts ranged from 13 to 155 neurons per hemisphere in Xenopus (n ⫽ 5), from 0 to 81 neurons per hemisphere in Discoglossus (n ⫽ 5), and from 16 to 112 neurons in Hyla (n ⫽ 5). In Bombina (n ⫽ 5), labeled cell bodies could be detected only in one animal (12 neurons per hemisphere). Neurons were found in both the dorsal and the ventral striatum. To investigate the distribution of immunoreactive neurons within the striatum, we divided it into three equal parts along its rostrocaudal axis: rostral, intermediate, and caudal striatum. In Xenopus, Discoglossus, and Hyla, significantly more enkephalinergic neurons were located in the rostral than in the intermediate or caudal striatum (Fig. 2). In the one Bombina with enkephalinimmunoreactive neurons, all labeled cells were found in the rostral and intermediate striatum.

Retrograde neurobiotin tracing studies Because we were interested in the exact boundaries of the anuran dorsal pallidum, we chose for tracer injections diencephalic areas that might be the target of pallidal projections (central and ventromedial thalamic nuclei). Because afferents and efferents of thalamic nuclei to brain areas other than the striatum are of no interest for the current investigation, we will describe only their connections with the striatum. Numbers of retrogradely labeled neurons were in general relatively low, because application sites were small (maximal diameter 40 –125 ␮m). Neurobiotin applications (Fig. 3B,C) into the central thalamic nucleus (n ⫽ 4 Hyla versicolor), the main input

Fig. 2. Leucine-enkephalin immunoreactivity in the striatum. A: Drawing indicating the position of B. B: Photomicrograph showing a sagittal section of the striatum of Xenopus laevis. Leucineenkephalin-immunoreactive neurons are indicated by arrowheads. C: First row: Raw numbers of immunoreactive neurons in the rostral, intermediate, and caudal striatum. Different animals are indicated by different symbols. Second row: Distribution of enkephalinimmunoreactive neurons in the rostral, intermediate, and caudal part of the striatum (mean ⫾ SD; 100% refers to total number of counted cells). *Number of labeled neurons in the intermediate and caudal striatum is significantly lower (P ⬍ 0.05, one-way ANOVA) compared with the rostral striatum. Scale bar ⫽ 100 ␮m.

area of the striatum, revealed numerous labeled fibers innervating the ipsilateral and to a lesser extent also the contralateral striatum. At caudal and intermediate striatal levels, central thalamic input fibers were confined to the lateral neuropil but terminated mainly within the area of cell bodies in the rostral striatum (Fig. 4). Retrogradely labeled neurons were concentrated in the most caudal part of the ipsilateral striatum, which stretches ventrally of the lateral amygdala to the rostral border of the anterior entopeduncular nucleus. In the rostral and intermediate part of the striatum, only a few neurons were found (Fig. 5, Table 1). Additionally, a few cells were labeled in the rostral aspects of the ipsilateral anterior entopeduncular nucleus. Tracer applications (Fig. 3A,B) into the ventromedial thalamic nucleus (n ⫽ 4 H. versicolor) labeled neurons in the ipsilateral intermediate and caudal striatum only. Those neurons were uniformly distributed over the dorsal and ventral part of the striatum (Fig. 6, Table 2). A few

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303 their numbers were nearly equal in both the dorsal and the ventral parts (Fig. 8). The proportion of double-labeled neurons was near zero in the rostral striatum but increased to values between 5.0% and 19.6% in the dorsal part of the intermediate striatum (Fig. 8). In the ventral part of the intermediate striatum, only 0.75– 6.6% of cells were doubly labeled.

DISCUSSION Methodological considerations The enkephalin immunohistochemical stainings revealed a high variability in the number of labeled striatal neurons among individuals. Total cell counts reached from zero to more than 100 immunoreactive neurons in the striatum of one hemisphere. This was not due to differences in the quality of staining; in all preparations, immunoreactive neurons of other brain areas had been darkly labeled on the same sections. Rather, there seem to be genuine interindividual differences concerning the number of neurons using enkephalin as a transmitter.

Comparison with other studies

Fig. 3. Neurobiotin injection sites into the central (C1– 4) and ventromedial (VM1– 4) thalamic nuclei of Hyla versicolor. A,B: Drawings of transverse sections with all injections. C: Photomicrograph of a tracer injection spot (arrow) in the central thalamic nucleus. Scale bars ⫽ 250 ␮m.

labeled fibers were present in the striatum only in the two animals with the largest injection sites. In the anterior entopeduncular nucleus, stained fibers were visible; a small number of retrogradely labeled neurons was found only in one animal.

Double-labeling tracing studies (n ⴝ 4) Application of OGDA (green fluorescent) into the lateral forebrain bundle at the level of the anterior thalamic nucleus labeled projection neurons throughout the entire striatum; their number was lowest in the rostral part of the striatum and steadily increased toward the caudal part (Fig. 7). The neurons were homogeneously distributed within each transverse section, showing no accumulation in the dorsal, ventral, superficial, or periventricular part. Retrograde labeling of the anterior entopeduncular nucleus could not be analyzed, because tracer applications in the lateral forebrain bundle were too close to this nucleus, and it was not possible to distinguish labeling caused by axonal tracer uptake from that caused by dendritic uptake. Application of TMRDA (red fluorescent) in the caudal striatum labeled numerous neurons in the rostral and intermediate striatum. These neurons, too, appeared homogeneously distributed on transverse sections. The number of labeled neurons was lowest in the rostral striatum and increased gradually toward the injection site. Comparing the distribution of neurons labeled by either TMRDA or OGDA revealed that, in the rostral striatum, the number of TMRDA neurons far exceeded the number of OGDA neurons, whereas, in the intermediate striatum,

Neurobiotin tracing studies. Qualitative evaluations of retrograde tracing experiments with application in the dorsal thalamus at intermediate levels of the rostrocaudal axis have been performed by other authors. Those injections resulted in retrogradely labeled neurons in the dorsal and ventral striatal subdivision (Marı´n et al., 1997b: Rana perezi, Xenopus laevis), but the distribution of neurons along the rostrocaudal axis has not been described. In two studies (Wilczynski and Northcutt, 1983: Rana catesbeiana; Hall and Feng, 1987: Rana pipiens), no retrogradely labeled neurons were found in the striatum, a result probably attributable to the transport characteristics of the HRP used in these studies. Immunohistochemistry. Enkephalin-immunoreactive neurons have already been described for the dorsal and ventral striatum of anurans (Merchenthaler, 1989: Rana esculenta). In the study by Marı´n et al. (1998b: Rana perezi), immunoreactivity of enkephalin was confined mainly to fibers and axon terminals. Quantitative analyses of labeled cell bodies have not been performed so far.

Criteria for defining the anuran dorsal striatopallidal system The definition of the dorsal striatum and the dorsal pallidum has been developed from mammals (see, e.g., Alexander and Crutcher, 1990; Graybiel, 1990a,b; Parent, 1990; Smith and Bolam, 1990; Holt et al., 1997; Nakano et al., 2000), and the corresponding structures in birds and reptiles have been named according to the mammalian model (Reiner et al., 1995; Marı´n et al., 1998a; Smeets et al., 2000). Immunohistochemically, the mammalian striatum (the “input structure of the basal ganglia”) is characterized by 90 –95% medium-sized spiny projection neurons immunoreactive for the neurotransmitter ␥-aminobutyric acid (GABA) and additionally for substance P or enkephalin. Hodologically, the striatum is defined by its glutamatergic inputs from different isocortical and dorsal thalamic regions (centromedian, parafascicular, and intralaminar nuclei) and its efferent projections to the external segment of the globus pallidus (indirect pathway, enkephalin im-

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Fig. 4. Photomicrographs of labeled structures in the striatum ipsilateral to the tracer injection into the central thalamic nucleus of Hyla versicolor. High magnifications from transverse sections, as indicated in D. A: Rostral striatum; labeled fibers are mainly concentrated in the periventricular layer of cell bodies. B: Intermediate

striatum; labeled fibers are distributed within the layer of the cell bodies and the neuropil. C: Caudal striatum; labeled fibers are mainly confined to the lateral neuropil. For abbreviations see list. Scale bars ⫽ 100 ␮m.

munoreactive) and to the internal segment of the globus pallidus and the substantia nigra pars reticulata (direct pathway, substance P immunoreactive). The mammalian pallidum (the “output structure of the basal ganglia”) contains large neurons immunoreactive for GABA and the calcium-binding protein parvalbumin. The neuropil is rich in substance P- and enkephalin-

immunoreactive (“wooly”) fibers (Haber and Nauta, 1983), but relatively poor in dopaminergic fibers and acetylcholinesterase. Hodologically, the dorsal pallidum is defined by its afferents from the striatum and efferents to the subthalamic nucleus (globus pallidus externus) and the ventral anterior and ventral lateral nuclei of the dorsal thalamus (globus pallidus internus).

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Fig. 5. A–E: Drawings of labeled structures in the striatum after tracer application into the central thalamic nucleus (arrow in E) of Hyla versicolor. Labeled fibers are indicated by short lines; labeled cell bodies are symbolized by triangles. The level of the transverse sections is indicated in the inset. For abbreviations see list. Scale bar ⫽ 500 ␮m. TABLE 1. Distribution of Retrogradely Labeled Neurons (Percentages) in the Striatum after Neurobiotin Injection Into the Central Thalamic Nucleus Animal C1 C2 C3 C4 Mean ⫾ SE

Rostral 1

Rostral 2

Interm. 1

Interm. 2

Caudal 1

Caudal 2

0 0 0 0 0.0 ⫾ 0.0

0 8 0 0 2.0 ⫾ 2.0

0 1 0 0 0.3 ⫾ 0.3

14 7 0 6 6.8 ⫾ 2.9

14 7 0 28 12.3 ⫾ 6.0

72 77 100 66 78.8 ⫾ 7.4

In birds, reptiles, and fishes, the globus pallidus is not subdivided into an internal and external segment; rather, the external and internal pallidal neurons seem to be fused into a single pallidal field (Reiner et al., 1998). We would therefore not expect a parcellation of striatum or dorsal pallidum in anuran amphibians. In contrast to the histological organization, the major neurotransmitter and neuropeptide systems seem to be highly conserved during the evolution of the basal ganglia; they have been found in birds, reptiles, and fishes as well. In general, this is also true for the connectivity of the basal ganglia (Medina and Reiner, 1995; Reiner et al., 1998; Marı´n et al., 1998a,c; Smeets et al., 2000). In the following paragraphs, we will therefore use hodological and immunohistological criteria of the mammalian basal ganglia to define the respective structures in anurans.

Thalamic tracer injections In the present study, tracer injections into the central thalamic nucleus (the main input area of the striatum) labeled thalamic input fibers, which course through the caudal striatum (the putative dorsal pallidum) to reach the rostral part (the putative striatum proper). Because

electron microscopic data are not yet available, we do not know how many of these axons are connected with caudal striatal neurons and how many are merely fibers of passage. Our tracing studies at least suggest that the thalamic input to the rostral striatum is stronger than that to the caudal striatum, matching the mammalian pattern: After tracer injections in the central thalamic nucleus, the anterogradely labeled fibers in the caudal striatum are confined to the lateral neuropil, whereas, in the rostral striatum, the fibers concentrate around the cell bodies. Even if all fibers in the caudal striatum synapse with striatal distal dendrites, their effect on the neuronal excitability would be low compared with that of the synapses on cell bodies and proximal dendrites of rostrostriatal neurons (Nieuwenhuys et al., 1998). Because of this strong input of dorsal thalamic fibers, the projection to the caudal striatum (see double-labeling studies), and the absence of neurons projecting to central and ventromedial thalamic nuclei, we suggest that the rostral striatum may be comparable to the striatum proper. After neurobiotin injections in the central and ventromedial thalamic nuclei, retrogradely labeled neurons were located mainly in the caudal part and, to a lesser extent, in

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Fig. 6. A–E: Drawings of labeled structures in the striatum after tracer application into the ventromedial thalamic nucleus (arrow in E) of Hyla versicolor. Labeled fibers are indicated by short lines; labeled cell bodies are symbolized by triangles. The level of the transverse sections is indicated in the inset. For abbreviations see list. Scale bar ⫽ 500 ␮m.

TABLE 2. Distribution of Retrogradely Labeled Neurons (Percentages) in the Striatum after Neurobiotin Injection Into the Ventromedial Thalamic Nucleus Animal VM1 VM2 VM3 VM4 Mean ⫾ SE

Rostral 1

Rostral 2

Interm. 1

Interm. 2

Caudal 1

Caudal 2

0 0 0 0 0.0 ⫾ 0.0

0 0 0 0 0.0 ⫾ 0.0

6 11 0 0 4.3 ⫾ 2.7

12 0 11 14 9.3 ⫾ 3.1

29 28 67 43 41.8 ⫾ 9.1

53 61 22 43 44.8 ⫾ 8.4

the intermediate part of the striatum. In the anterior entopeduncular nucleus, which was proposed to represent a part of the dorsal pallidum in amphibians (Marı´n et al., 1998b,c), a few projection neurons were found as well, but not in the same proportion as in the caudal striatum. The rostral striatum contains only a few projection neurons. We therefore suggest that, because of its projection to the thalamus, the intermediate and caudal striatum may be homologous to the dorsal pallidum. The existence or the location of a subthalamic nucleus, which is one of the major targets of the mammalian pallidum, is an unresolved issue so far. In birds, the anterior nucleus of the ansa lenticularis is comparable with the mammalian subthalamic nucleus (Jiao et al., 2000), whereas, in reptiles, the anterior entopeduncular nucleus has been considered homologous to the mammalian subthalamic nucleus (Medina and Reiner, 1995). From ontogenetic studies in amniotes, it is known that the subthalamic nucleus is located in the diencephalic prosomere 4 (Reiner et al., 1998), whereas the anuran anterior entopeduncular area develops from prosomere 5 (Puelles, 2001; Sua´rez et al., 2002). In reptiles, the anterior entopeduncular nucleus has been homologized with the mammalian

external part of the globus pallidus in a more recent study (Sua´rez et al., 2002). It therefore seems unlikely that the anuran anterior entopeduncular nucleus is homologous to the mammalian subthalamic nucleus. Further studies are necessary to delineate an anuran subthalamic nucleus, which then can serve as another criterion to define the anuran dorsal pallidum.

Double-labeling studies Application of OGDA in the lateral forebrain bundle resulted in retrogradely labeled neurons mainly in the intermediate and caudal part of the striatum. Because most of the striatofugal fibers travel within the lateral forebrain bundle, we can conclude that the intermediate and caudal subdivisions are the output structures of the anuran striatum. Application of TMRDA into the caudal striatum revealed that many neurons of the rostral and intermediate striatum project into the caudal part. These findings corroborate the hypothesis developed above comparing the anuran rostral striatum with the mammalian striatum proper and the anuran caudal striatum with the mammalian dorsal pallidum. Because of strong projections to both the caudal striatum and the lateral forebrain

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Fig. 7. Inverted laser scanning image of labeled structures in the intermediate striatum of Bombina orientalis (transverse section). Tracer injection site (Oregon green-conjugated dextran amine) was the ipsilateral forebrain bundle. Inset: Distribution of retrogradely

labeled cell bodies (mean values ⫾ SD) in the striatum. Each column represents three transverse sections (50 ␮m each). For abbreviations see list. Scale bar ⫽ 100 ␮m.

bundle/thalamic nuclei, the intermediate striatum seems to share features of the striatum proper and the dorsal pallidum. The double-labeling analysis demonstrated that the population of rostral and intermediate striatal neurons projecting into the lateral forebrain bundle and that innervating the caudal striatum are completely separated, except in the dorsal part of the intermediate striatum. Here, up to 20% of neurons send axon collaterals into both the caudal striatum and the lateral forebrain bundle. Provided that the anuran rostral striatum is comparable with the mammalian striatum proper, and the anuran caudal striatum with the mammalian globus pallidus, this separation of striatal descending projections may be a first clue to the existence of a direct and indirect striatal output pathway in amphibians. On the other hand, the doublelabeled neurons in the intermediate striatum may be comparable to the mammalian striatal neurons projecting

both via the direct pathway (to the globus pallidus internus and the substantia nigra pars reticulata) and via the indirect pathway (to the globus pallidus externus; Wu et al., 2000; Parent et al., 2000).

Immunohistochemistry Our enkephalin immunostainings in several anuran species have demonstrated that more than 90% of neurons immunoreactive for enkephalin are located in the rostral and intermediate striatum in all animals, with one exception (in Discoglossus pictus), in which enkephalinergic neurons were distributed equally throughout the striatum. In the striatum of mammals, approximately 90% of all neurons contain either enkephalin or substance P (Graybiel, 1990b), whereas the dorsal pallidum was thought to be devoid of such neurons. However, a recent study in mammals has shown that 40% of pallidal neurons also express preproenkephalin (Voorn et al., 1999), but the

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Fig. 8. Laser scanning images of retrogradely labeled neurons in the intermediate striatum (transverse section; see inset) of Bombina orientalis after application of tetramethyl rhodamine-conjugated dextran amine (A) in the caudal striatum and application of Oregon green-conjugated dextran amine (B) in the lateral forebrain bundle. C shows the overlay of images A and B, revealing a double-labeled

H. ENDEPOLS ET AL.

neuron (asterisk). Graphs in the right column indicate the distribution of TRDA- and OGDA-labeled neurons (mean values) in the dorsal and ventral part of the rostral and intermediate striatum. Note that double-labeled neurons are located mainly in the dorsal part of the intermediate striatum. For abbreviations see list. Scale bar ⫽ 20 ␮m.

ANURAN DORSAL STRIATOPALLIDAL SYSTEM intensity of labeling of the pallidal neurons is considerably lower than that of the striatal neurons. Hence, our immunostainings exactly match the mammalian pattern and therefore support the hypothesis that the anuran rostral striatum is homologous to the mammalian striatum, whereas the caudal striatum represents the dorsal pallidum.

CONCLUSIONS We propose, on the basis of our hodological data, the following theory of neural processing in the anuran striatum: Although the caudal and intermediate striatum most likely receives ascending projections as well, the rostral striatum is the main input area of the anuran basal ganglia. Information enters the striatum in its rostral aspects and is processed in neurons that project caudalward. There it reaches an increasing number of projection neurons in the dorsal and ventral subdivisions of the intermediate and caudal striatum, which give rise to descending projections. Quantitative analysis of retrogradely labeled neurons confirm that projection neurons are located mainly in the intermediate and caudal striatum, in both dorsal and ventral subdivisions. Hodological as well as immunohistochemical data are consistent with the hypothesis that the anuran rostral striatum is homologous to the mammalian striatum proper, whereas the anuran caudal striatum (ventral and dorsal part) is homologous to the mammalian dorsal pallidum. A sharp border between the two nuclei, as it is the case in mammals, is not found, but a relatively large transition area represented by the intermediate striatum can be observed. The anuran dorsal pallidum as defined here contains the whole dorsal pallidum described by Reiner and Medina (1995) in the superficial part of the caudal striatum and also a part of the respective structure (the ventromedial part of the caudal striatum) described by Marı´n et al. (1998b,c; Fig. 1). With the data available, we cannot decide whether the anterior entopeduncular nucleus is part of the dorsal pallidum as well. To delineate the caudal margins of the dorsal pallidum, additional work (e.g., the definition of the anuran subthalamic nucleus) is needed.

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