An indirect basal ganglia pathway in anuran amphibians?

Journal of Chemical Neuroanatomy 40 (2010) 21–35

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An indirect basal ganglia pathway in anuran amphibians? Silke Maier a, Wolfgang Walkowiak a, Harald Luksch a,b, Heike Endepols a,c,* a

Institute of Zoology, University of Cologne, Weyertal 119, 50923 Ko¨ln, Germany Institute of Zoology, Technische Universita¨t Mu¨nchen, Liesel Beckmann-Straße 4, 85350 Freising-Weihenstephan, Germany c Max Planck Institute for Neurological Research, Gleueler Str. 50, 50931 Ko¨ln, Germany b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 December 2009 Received in revised form 24 February 2010 Accepted 24 February 2010 Available online 4 March 2010

The mammalian subthalamic nucleus (STN) is a glutamatergic cell group within the indirect pathway of the basal ganglia. It receives input from the external globus pallidus (GP) and in turn projects to the internal GP and the substantia nigra pars reticulata (SNr). While the direct pathway from striatum to SNr is well established in anurans, it is unknown whether they possess an indirect pathway including a STN homologue. The subthalamic region comprises the dorsocaudal suprachiasmatic nucleus (dcSC), the posterior entopeduncular nucleus (EP), and the ventral part of the ventral thalamus (vVM/VL). In the firebellied toad Bombina orientalis we investigated whether one of these areas match the criteria established for the mammalian STN. We delineated the SNr in the midbrain tegmentum by labeling the striatonigral terminal field by means of GABA-, substance P-, and enkephalin immunohistochemistry and striatal tracer injections. Subsequently, we used double fluorescence tracing with injections into the SNr and GP to stain different parts of the indirect pathway. Confocal laser scan analysis revealed that dcSC, EP, and vVM/VL contain retrogradely labeled neurons projecting to the SNr, contacted by anterogradely labeled terminals arising in the GP. Immunohistochemical stainings with antibodies against glutamate and the glutamate transporters EAAC1 and vGluT2 demonstrated that the investigated nuclei contain glutamatergic neurons. Our results suggest that all regions in the subthalamic region fulfill our morphological criteria, except the connection back to the GP. An indirect basal ganglia pathway seems to be present in anuran amphibians, although we cannot exclusively delineate an STN homologue. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Subthalamic nucleus Substantia nigra Striatum Globus pallidus Homology

Abbreviations: I–V, layer I–V of the optic tectum; VI, layer VI of the optic tectum; VII– IX, layer VII–IX of the optic tectum; A, anterior thalamic nucleus; Ad, anterodorsal tegmental nucleus; Av, anteroventral tegmental nucleus; B, nucleus of Bellonci; C, central thalamic nucleus; CA/BNST, central amygdala/bed nucleus of the stria terminalis; cPrm, caudal part of the nucleus profundus mesencephali; cStr, caudal striatum (=globus pallidus); D, direct pathway; DB, diagonal band of Broca; DcSC, dorsocaudal suprachiasmatic nucleus; DSNr, dorsal part of the substantia nigra pars reticulata; EAAC1, excitatory amino acid carrier-1; EP, posterior entopeduncular nucleus; GABA, gamma-aminobutyric acid; GP, globus pallidus; Hyp, hypothalamus; i, indirect pathway; Isth, nucleus isthmi; La, anterolateral thalamic nucleus; Lfb, lateral forebrain bundle; LH, lateral nucleus of the hypothalamus; lPal, lateral pallium; MPal, medial pallium; N.III, nucleus of the oculomotor nerve; NA, nucleus accumbens; P, posterior thalamic nucleus; Pd, posterodorsal tegmental nucleus; PreT, pretectum; Pv, posteroventral tegmental nucleus; RStr, rostral striatum (=striatum proper); SC, suprachiasmatic nucleus; Sd, dorsal septum; SIR, superficial isthmal reticular nucleus; Sl, lateral septum; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; Str, striatum; Tec, optic tectum; Teg, tegmentum; Tl, laminar nucleus of the torus semicircularis; Tm, magnocellular nucleus of the torus semicircularis; Tp, principal nucleus of the torus semicircularis; TP, posterior tuberculum; vGluT2, vesicular glutamate transporter 2; VH, ventral hypothalamic nucleus; VL, ventrolateral thalamic nucleus; VLd, dorsal part of the ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; vPal, ventral pallidum; vSNr, ventral part of the substantia nigra pars reticulata; vVM/VL, ventral part of the VM/VL; VTh, ventral thalamus (=VM + VL). * Corresponding author at: Max Planck Institute for Neurological Research, Gleueler Str. 50, 50931 Ko¨ln, Germany. Tel.: +49 221 4726 227; fax: +49 221 4726 298. E-mail address: [email protected] (H. Endepols). 0891-0618/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2010.02.004

1. Introduction The basal ganglia and their associated structures form an evolutionary conserved system with anatomical features shared by all vertebrate groups (Reiner et al., 1984, 1998; Parent, 1986; Russchen et al., 1987a,b; Medina and Reiner, 1995; Marı´n et al., 1998a,b; Smeets et al., 2000; Gonza´lez et al., 2002; Endepols et al., 2004). In anuran amphibians, homologues of the striatum, dorsal and ventral pallidum, substantia nigra pars compacta and pars reticulata have been described, and there is evidence that anurans possess at least the direct pathway of the basal ganglia loops (Marı´n et al., 1997a,b, 1998a; Endepols et al., 2004). Whether an indirect pathway exists as well has not been investigated so far and will be the rationale of this study. In the mammalian brain, the subthalamic nucleus (STN) is an important part of the indirect pathway of the basal ganglia. It is embedded in the motor, associative, and limbic loops and hence modulates not only motor, but also cognitive and affective processes (Temel et al., 2005). The STN receives afferents from the cortex, globus pallidus, parafascicular and centromedian nuclei of the thalamus, substantia nigra pars compacta, pedunculopontine nucleus, and dorsal raphe nuclei. Efferents are entirely

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S. Maier et al. / Journal of Chemical Neuroanatomy 40 (2010) 21–35

glutamatergic and directed towards the globus pallidus, substantia nigra pars reticulata and pars compacta, and the pedunculopontine nucleus (for review see Hamani et al., 2004; Temel et al., 2005; Tan et al., 2006). It has been suggested that the indirect basal ganglia pathway inhibits unwanted and/or conflicting behavioral alternatives while the direct pathway facilitates planned actions. This may be achieved by an antagonistic balance between the two pathways, or in a contrast enhancing center-surround fashion (for review see Gale et al., 2008). It is therefore not surprising that indirect basal ganglia pathways have been found in other vertebrate groups capable of planned actions and executive control. Homologues of the STN have been discovered in birds and reptiles, defined by their location in the diencephalic prosomere 4 (Puelles and Medina, 1994; Jiao et al., 2000) and by their connections, transmitters and receptors similar to their mammalian counterparts. Homologues of the mammalian STN are the STN in birds, formerly called anterior nucleus of the ansa lenticularis (Jiao et al., 2000; Reiner et al., 2004), and the anterior entopeduncular nucleus in reptiles (Fowler et al., 1999; Medina and Reiner, 1995). In anuran amphibians, however, there seemed to be no need for an indirect basal ganglia pathway, since their behaviors have been regarded as non-volitional and genetically determined. However, conceptual advances over the past decades have gone beyond such simple attribution of functions to structures in the vertebrate brain, and the suggested absence of behavioral complexity in anurans would not imply the absence of specific brain pathways. Furthermore, recent studies have demonstrated that there is enough behavioral variability for frogs to decide between strategies, leaving room for individual behavior. An example from the context of anuran mating behavior is that male frogs can decide to call in order to attract females, or take the role of a silent ‘‘satellite male’’ trying to mate with females which are on their way to a nearby calling male (Lucas et al., 1996). Also, males can change their calling strategy immediately as a response to the behavior (calling/not calling) of neighboring males (Schwartz et al., 2002), and are able to adjust their amount of aggression according to the fact whether the nearby conspecific rivals are familiar to them or not (Bee and Gerhardt, 2002). In female mating behavior, phonotaxis and preferences for certain calling males can be variable as well (Grafe, 1999). We have thus reassessed the evidence of an indirect basal ganglia pathway in anuran amphibians, on the basis of criteria derived from ‘‘higher’’ vertebrate groups. In order to define a STN in anurans, we looked for a brain area that (1) contains glutamatergic neurons, (2) displays the characteristic connectional patterns of the indirect basal ganglia pathways: reciprocal connections with the globus pallidus and efferents to the substantia nigra pars reticulata, and (3) is located in prosomere 4. Our region of interest (i.e. subthalamic/prosomere 4 region) is comprised of the posterior entopeduncular nucleus (EP), the dorsocaudal suprachiasmatic nucleus (dcSC), and the ventral part of the ventral thalamic nuclei (vVM/VL; not related to the mammalian motor thalamus VM/VL). Here we present hodological and immunohistochemical evidence suggesting that an indirect pathway as well as a STN homologue exists in anuran amphibians.

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2. Materials and methods For this study we used a total of 39 adult Bombina orientalis, both sexes, from our own breeding stock. All experiments comply with Principles of Animal Care, publication no 86-23, revised 1985, of the National Institutes of Health and also with the current German laws. 2.1. Antibodies Monoclonal anti-glutamate (mouse IgG1 isotype; Sigma, G 9282) was produced from mouse GLU-4 hybridoma with L-glutamic acid conjugated to keyhole limpet hemocyanin as immunogen. Specificity was tested using indirect and competitive ELISA. A weak cross-reaction was observed with conjugated Dglutamate, L-glutamine, L-aspartate, D-aspartate, L-asparagine, b-alanine, glycine, 5-amino valeric acid, GABA, and glycyl-aspartate. Reactivity with frog glutamate has not been confirmed yet for this antibody, although glutamate immunoreactivity has been demonstrated in anuran brains before (e.g. Reichenberger et al., 1997). Monoclonal anti-neuronal glutamate transporter EAAC1 (clone 4D6.2; mouse IgG1; Chemicon, MAB1587) was raised against the C-terminus peptide (amino acid 510–524). In amphibians, EAAC1 has been demonstrated in the retina (Schultz and Stell, 1996; Zhao and Yang, 2001), but other parts of the brain have not been examined. Reactivity of our antibody with frog neuronal glutamate transporter has not been confirmed yet. Polyclonal anti-vesicular glutamate transporter 2 (anti-vGluT2, rabbit affinity purified IgG1, Alpha Diagnostic, VGLUT22-A) was raised against the 20 amino acid synthetic peptide from rat, conjugated to keyhole limpet hemocyanin. Specificity and cross-reactivity were not tested. Monoclonal anti-GABA (mouse IgG; Swant, MAB 3A12) was raised against GABA conjugated to BSA. Specificity was tested using ELISA (Matute and Streit, 1986). A very weak cross-reaction occurred with b-alanine, glycine, and glutamate. No cross-reaction was observed with aspartate and taurine. Reactivity with frog GABA has been demonstrated (Reichenberger et al., 1997). The monoclonal mouse anti-tyrosine hydroxylase IgG1k antibody (clone LNC1; Chemicon, MAB318) was raised against purified tyrosine hydroxylase from PC12 cells. It recognizes an epitope outside of the N terminus, labels a band at 59–61 kDa in Western blots, and does not cross-react with dopamine hydroxylase, phenylalanine hydroxylase, tryptophan hydroxylase, dihydropteridine reductase, sepiapterin reductase, or phenethanolamine-N-methyl transferase. Reactivity with frog tyrosine hydroxylase has been confirmed (Chu and Wilczynski, 2002). Monoclonal anti-enkephalin (mouse IgG; Chemicon, MAB350) was raised against Leu5-enkephalin conjugated to BSA and recognizes Met5- and Leu5enkephalin. It displays about 40% cross-reactivity with C-terminal extended Metenkephalin hexapeptides and 7% cross-reactivity with the extended heptapeptide (Arg-Phe-OH), but does not recognize other endogenous peptides. There is no crossreactivity to beta-endorphin or dynorphin. The amino acid sequences of Met5- and Leu5-enkephalin are the same throughout vertebrate groups (Dores et al., 2000; Salzet, 2001), so reactivity of this antibody with rat enkephalin (Freeman et al., 2003) predicts reactivity in the frog as well, but has not been confirmed so far. Polyclonal anti-substance P (IBL, MI60/370) was raised in rabbit against synthetic substance P (code 7451) conjugated to BSA. It cross-reacts weakly with bombesin, but does not recognize neurokinin A and B, kassinin, eledoisin, physalaemin, neuromedin B and C. Generally, antibodies against mammalian substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2) recognize amphibian substance P-like peptides (e.g. ranakinin: Lys-Pro-Asn-Pro-Glu-Arg-PheTyr-Gly-Leu-MetNH2 or bufokinin: Lys-Pro-Arg-Pro-Glu-Arg-Phe-Tyr-Gly-LeuMetNH2; O’Harte et al., 1991; Moreno and Gonza´lez, 2007) as well. Reactivity of our antibody in frogs, including preadsorption controls, has been tested elsewhere (Schmidt et al., 1989). 2.2. Immunohistochemical procedures Frogs were anaesthetised with 0.2% tricaine–methansulfonate-solution (MS 222, Sigma–Aldrich, USA; Ohr, 1976; Luksch et al., 1996), cooled down to body temperature of 5 8C and perfused transcardially with 40 ml ice-cold oxygenated Ringer’s solution (Straka and Dieringer, 1993). The brain was removed from the skull and fixed overnight with 4% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M sodium phosphate buffer (PB) pH 7.4. After washing in 0.05 M Tris with sodium bisulfite (TrisMBS) the brains were dehydrated in a 15% sucrose solution.

Fig. 1. Immunohistochemistry and anterograde tracing in the midbrain tegmentum and diencephalon. (A) Photomicrograph of enkephalin immunoreactivity, showing the dorsal (arrows) and the ventral (arrowheads) terminal field in the anterior tegmentum. (B) GABA immunoreactivity labeling the dorsal (arrows) and ventral (arrowheads) fiber plexus in the anterior tegmentum as well as immunoreactive neurons in the posterior tegmentum (insert). (C) Anterograde labeling of striatonigral projections after a small injection in the rostral striatum, following the same pattern as enkephalinergic and GABAergic fibers. (D) Substance P immunoreactivity in the anterior tegmentum. (E) Tyrosine hydroxylase immunoreactive neurons in the interpeduncular nucleus (VTA homologue). (F) Tyrosine hydroxylase immunoreactivity in the diencephalic posterior tuberculum, the dorsal part of which is homologous to the SNc. (G–J) Schematic drawing of GABA-immunoreactive neurons (right side) and congruent enkephalinergic, substance P- and GABAergic fibers (left side) which are used to define the anuran SNr. The frame in G indicates the localization of E, the frame in I indicates the localization of A–D. The rostrocaudal level of F corresponds to Fig. 8B. Scale bars: 50 mm (A–D), 20 mm (insert in B), 150 mm (E, F).

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The brains were embedded in tissue freezing medium, frozen with liquid nitrogen and cut into 25 mm transverse sections using a cryostat (2800 Frigocut, Germany). Sections were directly transferred onto acidulated slides and dried at 37 C. The sections were encircled with a hydrophobic PAP-pen (Science Services, Germany) and rinsed in TrisMBS (3 10 min). To block non-specific binding sites the sections were covered with 1% normal horse serum (NHS, Vectastain ABC mouse kit; USA) in TrisMBS for 1 h at room temperature. The blocking serum was removed and without rinsing, sections were covered with the primary antibody (anti-glutamate, n = 3; anti-EAAC1 [excitatory amino acid carrier-1], n = 3), diluted 1:400 in TrisMBS containing 0.5% Triton X-100 and 0.5% NHS. After 24 h the slides were washed in TrisMBS for 10 min and subsequently in 0.05 M Tris with NaCl (TBS 2 10 min). The secondary antibody solution (biotinylated anti-mouse IgG; Vectastain ABC mouse kit, Vector, USA), diluted 1:100 in TBS containing 1% NHS, was applied to the slides and incubated for 1 h. The slides were first washed in TBS for 10 min and then twice in 0.05 M phosphate buffer with NaCl (PBS) for 10 min. The sections were incubated for 1 h in avidin–biotin-HRP solution (Vectastain ABC mouse kit, USA; 1% in PBS). The slides were then rinsed for 10 min in PBS and afterwards twice in PB. HRP was visualized with the chromogen diaminobenzidine (DAB, Boehringer Ingelheim, Germany) and heavy metal intensification following the protocol of Adams (1981). H2O2 was provided by a glucose oxidase reaction (Shu et al., 1988). After staining, slides were dehydrated in alcohol and coverslipped with Corbit (Hecht, Germany). Enkephalin, substance P, vGluT2, and tyrosine hydroxylase immunohistochemistry (n = 3 each) followed the same protocol, only with PB instead of Tris and TrisMBS. The Met + Leu-enkephalin antibody was diluted 1:300, the substance P antibody was diluted 1:2000, the vGluT2 antibody was diluted 1:400, and the tyrosine hydroxylase antibody was diluted 1:400. The secondary antibody for substance P and vGluT2 staining was biotinylated anti-rabbit IgG (Vectastain ABC rabbit kit), diluted 1:100. In control stainings, either the primary or the secondary antibody was omitted. For glutamate, EAAC1, and vGluT2, additional preadsorption controls were done. For EAAC1 and vGluT2 controls, the antibody was preadsorbed with EAAC1 control peptide (Chemicon) or vGluT2 control peptide (Alpha Diagnostic), respectively, diluted 1:10 in the primary antibody solution. For glutamate preadsorption controls, glutamate (1 mg) was coupled to keyhole limpet hemocyanin (KLH; 1.74 ml; Sigma) using glutaraldehyde (7 ml). The KLH–glutamate–glutaraldehyde complex was then added to the primary antibody solution. For GABA-immunohistochemistry, frogs (n = 4) were deeply anesthetized in 0.2% MS 222 for 10 min and cooled down to a body temperature of 5 8C. Subsequently, they were perfused transcardially with 10 ml of 2% paraformaldehyde in sodium acetate buffer, pH 6.5, followed by 10 ml of 2% paraformaldehyde +0.5% glutaraldehyde in sodium borate buffer, pH 8.5 (protocol adapted from Sloviter et al., 1996). The brains were removed from the skull and were immersed overnight in the sodium borate fixative at 4 8C. After a standard embedding procedure, brains were cut into two or three series of 25 mm thick transverse or sagittal sections on a cryostat (Reichert-Jung, Frigocut 2800). The sections were then immersed in the primary antibody solution (1:300 in TRIS buffer) and rocked gently for 72 h at room temperature. After washing for several hours in TRIS, sections were incubated in the secondary antibody solution (anti-mouse IgG; Vectastain ABC mouse kit) overnight at room temperature. After rinsing for several hours, the binding sites were visualized by means of an avidin–biotin–horseradish peroxidase complex (Vectastain ABC mouse kit; 2 h at room temperature) and DAB (see above). Sections were rinsed three times in 0.1 M PB, pH 7.4, dehydrated in ethanol, cleared in xylene and mounted in Corbit. In control experiments, either the primary or the secondary antibody was omitted. As an additional control, the primary antibody was preadsorbed with GABA (200 mM). The stained sections were examined with a microscope (Leica DM LB, Germany). Telencephalic areas were named according to Endepols et al. (2004), the diencephalic areas after Neary and Northcutt (1983) and the other brain areas after Ten Donkelaar (1998).

2.3. Fluorescence tracing Three frogs were used for double fluorescent labeling. We applied red fluorescent dextran-tetramethylrhodamine (MW 10,000; Molecular Probes) to the caudal striatum (homologous to the globus pallidus) and green fluorescent dextran-Alexa Fluor 488 (MW 10,000; Molecular Probes) to the SNr in the tegmentum. Prior to application, the tracer was dissolved in distilled water and recrystallized at the tip of a glass micropipette. Using a micromanipulator, the micropipette holding the tracer crystal was advanced into striatum or tegmentum, using a lateral approach. During retraction of the micropipette, the tracer crystal detached and remained inside the brain tissue. After the application the brains were stored in Ringer’s solution at 4 8C for 2 days to allow uptake and transportation of the tracer. The brains were fixed over night in 0.1 M PB containing 4% paraformaldehyde and 1.25% glutaraldehyde. After washing in PB the brains were embedded in tissue freezing medium and cut into 25 mm transverse sections with a cryostatic microtome. After washing several times in PB the slides were transferred to a sodiumborohydride bath (0.25% in PB) to block autofluorescence of glutaraldehyde. The slides were then coverslipped with glycerolgelatine and examined with a confocal laser scan microscope (Zeiss LSM 510).

2.4. Neurobiotin tracing The tracer biotin ethylenediamine (10% in 0.3 M sodium acetate solution; Neurobiotin, Molecular Probes, USA) was injected iontophoretically into the midbrain tegmentum (n = 11) by applying positive current (250 nA for 30 min). Control injections were done in the striatum (n = 3), the globus pallidus (n = 2) and the dorsal thalamus/pretectum (n = 1). The brains were incubated for 48 h at 4 8C in Ringer’s solution and afterwards fixed with 4% paraformaldehyde and 1.25% glutaraldehyde in PB overnight. After washing in PB, they were embedded in 4% agar (Merck, Germany) in PB and cut into 50 mm transverse sections using a vibratome (Microslicer DTK-3000, Japan). Sections were directly mounted onto chrome alum/gelatine-coated slides, dried at 37 C, rinsed in PB for 10 min, and incubated with 2% streptavidin–HRP (Amersham Biosciences, UK) +0.5% Triton X100 in PB overnight. After several washes in PB, HRP was visualized with DAB (see above). After staining, sections were dehydrated with alcohol and coverslipped with Corbit.

3. Results Immunohistochemical stainings and tracing experiments labeled neurons and fibers all over the anuran brain. Since a survey of all labeled structures is beyond the scope of this article, we will only report results which concern the definition of the anuran indirect pathway. 3.1. Enkephalin-, substance P-, and GABA-immunohistochemistry of the tegmentum While only very few cells were labeled in the dorsal and ventral tegmental nuclei, enkephalin immunohistochemistry strongly stained numerous fibers throughout the tegmentum. Two conspicuous fiber plexus were found: one in the transition zone between anterodorsal and anteroventral nucleus, the other in the neuropil lateral to the dorsal part of the anteroventral and posteroventral nuclei (Fig. 1A, G–J). Substance P immunohistochemistry resulted in exactly the same pattern (Fig. 1D, G–J). GABA-immunohistochemistry yielded two labeled terminal fields identical to the ones described with the enkephalin antibody (Fig. 1B). Labeled cell bodies were found in the anterior as well as the posterior tegmentum (Fig. 1B[insert], G–J and supplementary Fig. 1). A prominent band of neurons was located in the transition area between dorsal and ventral posterior tegmental nucleus (Fig. 1J), but did not overlap with the more rostrally situated GABA-/enkephalin-immunoreactive terminal field. 3.2. Tyrosine hydroxylase staining of the SNc/VTA complex Tyrosine hydroxylase immunohistochemistry was performed to confirm that the SNc/VTA complex of Bombina is in the same location as described for Xenopus laevis and Rana perezi (Marı´n et al., 1997d). Indeed, immunoreactive neurons were found in the interpeduncular nucleus (Figs. 1 and 9Figs. 1E and 9C) and the posterior tubercle (Figs. 1 and 9Figs. 1F and 9B). Although the main terminal field of striatal axons (see below) began at the same rostrocaudal level where interpeduncular neurons were located (Fig. 1G), it did not touch the short, ventrally directed interpeduncular dendrites. 3.3. Neurobiotin tracing in the striatum Tracer injection into the striatum revealed a strong projection to the midbrain. Striatal fibers traveled within the lateral forebrain bundle and formed a dense fiber plexus in the lateral neuropil of the ventral tegmental nuclei (Fig. 1C). From there, fibers entered the cell layers of the ventral tegmentum. Another fiber plexus was found in the transition zone between the anterodorsal and anteroventral tegmental nuclei. This terminal field overlapped exactly with the dorsal fiber plexus seen after enkephalin, substance P and GABA-immunohistochemistry (Fig. 1A, B and D). Retrogradely labeled cells were not found in the tegmentum.

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3.4. Neurobiotin tracing in the globus pallidus Tracer injection into the globus pallidus labeled numerous fibers in the lateral forebrain bundle on their way to the midbrain. In the tegmentum, we found the same fiber plexus in the lateral neuropil and the cellular zone of the ventral tegmental nuclei as after striatal injections (Fig. 2C and D). However, a terminal field in the transition zone between anterodorsal and anteroventral tegmentum was not labeled after pallidal injections. In the subthalamic area, fibers were found in the EP, the dcSC, VM, and VL (Fig. 2B). 3.5. Glutamate-/EAAC1/vGluT2- and GABA-immunohistochemistry of the subthalamic region The EAAC1 (=excitatory amino acid carrier-1) antibody labeled the cytoplasm of cell bodies and proximal dendrites. Cell nuclei, distal dendrites, and axons were not labeled. In the EP, dcSC, and the VM/VL many neurons were darkly stained (Figs. 3A and 4A,B). Similar to the EAAC1 antibody, the glutamate and vGluT2 antiserum labeled cell bodies and proximal

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dendrites. Both antibodies stained cells in the EP, dcSC, and VL, while in the VM immunopositive neurons were seen with vGluT2 only (Figs. 3B,C and 4C–F). GABA-immunohistochemistry revealed that the EP and the VM were devoid of labeled neurons. Only in the VL and dcSC, some scattered cells were stained (Figs. 3D and 4G,H). 3.6. Neurobiotin tracing in the dorsal thalamus/pretectum This tracer injection involved an area immediately caudal to the habenula, including the pretectum (prosomere 1; see Mila´n and Puelles, 2000; Brox et al., 2003; Morona and Gonza´lez, 2008), and labeled numerous neuronal cell bodies in the EP (Fig. 5). After injections directly into the habenula, however, EP neurons were not stained (not shown). 3.7. Neurobiotin tracing in the tegmentum Fig. 6 shows all injection sites that resulted in strong retrograde staining of neurons in the striatum (n = 11), indicating that we have labeled fibers of the direct pathway. Nearly all

Fig. 2. Photomicrographs of neurobiotin tracing with an injection in the globus pallidus (arrow in A). (B) Anterogradely labeled fibers in the subthalamic region. Soma-like structures in the SC and in the hypothalamic region below the SC are not retrogradely labeled neurons, but neuromelanin granulae filling the cell bodies of many cells. In situ, the brownish neuromelanin is easily distinguished from black neurobiotin labeling. (C and D) Anterogradely labeled fibers in the anterior (C) and posterior (D) tegmentum. Scale bar: 150 mm.

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Fig. 3. Overview of immunohistochemistry in the diencephalon at the rostrocaudal level of the subthalamic region. Scale bar for A–D: 5 mm.

injections stained neurons in the dcSC, VM, and VL. In the EP neurons were only labeled when the injection touched the Ad/Av transition zone (Fig. 7). Table 1 summarizes retrograde labeling in the striatum and the subthalamic region after tegmental tracer injections. 3.8. Double fluorescence tracing After injection of green dextran-Alexa Fluor 488 into the dorsal tegmental area retrogradely labeled neurons were found in the EP, dcSC, and vVM/VL. Dendrites of EP neurons (Fig. 8) were found to course radially through the lateral forebrain bundle. Injection of red dextran-tetramethylrhodamine into the dorsal pallidum yielded numerous anterogradely labeled axons, descending into the di- and mesencephalon. Most of those fibers traveled within the lateral forebrain bundle and contacted green-labeled EP neurons. Other red-labeled pallidal axons reached the dcSC and were found in close association with green-labeled dcSC and ventral thalamic neurons. Red-labeled neurons in the subthalamic region were rare.

Table 1 Retrograde labeling of cell bodies in the subthalamic region after neurobiotin injection in the tegmentum. Animal

Injection site

Striatum

dSC

Bo1 Bo2 Bo3 Bo4 Bo5 Bo6 Bo7 Bo8 Bo9 Bo10 Bo11

rostral Av, neuropil Av, neuropil Av rostral Ad, neuropil Pv T Pd/Pv Pv Ad rostral Av Av T Ad/Av

++ ++ ++ +++ ++ ++ ++ ++ ++ +++ +++

(+) + + ++ ++ (+) + ++ +++ ++ ++

T Ad/Av = transition zone between Ad and Av. T Pd/Pv = transition zone between Pd and Pv. (+): 1 neuron. +: 1–5 neurons. ++: 5–20 neurons. +++: more than 20 neurons.

EP

++

+ + (+) +

vVM

vVL

? ? + ++ +

? ?

(+) ++ +++ ++ ++

++ + (+) (+) ++ +++ ++ ++

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Fig. 4. Immunohistochemistry in the subthalamic region. Left column: EP; middle column: vVM/VL and dcSC; right column: schematic drawing of hemisections with locations of immunopositive neurons. (A and B) EAAC1; (C and D) glutamate; (E and F) vGluT2; (G and H) GABA. Scale bar (for A–H): 75 mm

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Fig. 5. Photomicrographs of neurobiotin labeled structures after tracer injection into the dorsal part of the anterior thalamic nucleus (arrow in A). Numerous neurons are retrogradely labeled in the posterior entopeduncular nucleus (A and B). (C) Detail from B. Scale bars: 200 mm in A and B, 100 mm in C.

4. Discussion 4.1. Methodological considerations 4.1.1. Immunohistochemistry In order to determine whether neurons of the EP, dcSC, and vVM/VL of Bombina orientalis are glutamatergic, we used

glutamate, EAAC1, vGluT2 and GABA-immunohistochemistry. The latter was also a means to identify the substantia nigra pars reticulata, which contains GABAergic neurons in mammals. Glutamate immunostainings alone are not sufficient to identify glutamatergic neurons because glutamate is a precursor of GABA and therefore present in GABAergic neurons as well. Additionally, we used an antibody against the neuronal glutamate transporter

Fig. 6. Neurobiotin injection sites into the midbrain tegmentum. The different patterns provide better visibility in overlapping injections. Level of sections corresponds to G, I, and J in Fig. 1.

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Fig. 7. Neurobiotin tracing after injection into the dorsal SNr. (A) Retrogradely labeled neurons in the striatum. (B) Retrogradely labeled neurons in the globus pallidus. (C) Labeled fibers and neurons in the diencephalon including the subthalamic region. (D) Injection site in the Ad/Av transition zone (arrow). (E) Subthalamic region immediately caudal of C, demonstrating more labeled neurons in the EP. Scale bars: 250 mm in D (valid for A–D), 100 mm in E.

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Fig. 8. Confocal laser scan images from the subthalamic region after tracer injection into the globus pallidus (red) and the dorsal SNr (green). (A) Posterior entopeduncular nucleus. (B) Detail from A, showing a retrogradely labeled EP neuron (green; projecting to the SNr) surrounded by fibers arising in the globus pallidus (red). (C) Image at the same XY-coordinates as in B, but 5.1 mm difference in Z-level. The asterisk indicates the neuronal cell body visible in B, and the arrows point at a basal dendrite (green) covered by pallidal presynaptic elements (red). (D) Neuron from the dcSC (double labeled, i.e. projecting to the SNr and the globus pallidus) surrounded by fibers from the globus pallidus (red). (E) Detail from D. (F) Whole subthalamic region with green-labeled structures connected to the SNr, and red-labeled structures connected to the globus pallidus. (G) Schematic drawing of the indirect pathway of Bombina orientalis. Scale bars: 50 mm (A and D), 10 mm (B, C and E), 100 mm (F). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

EAAC1 and the vesicular glutamate transporter vGluT2, which labels mainly glutamatergic neurons. However, EAAC1 can also be expressed in GABAergic cells (Coco et al., 1997; Conti et al., 1998; Sepkuty et al., 2002). If our glutamate-ir neurons were in fact GABAergic and used the cytosolic glutamate as a precursor for GABA, GABA should be detectable in the soma as well. Our stainings revealed that the EP, vVM/VL, and dcSC contained numerous glutamate-, vGluT2-, and EAAC1-immunoreactive

neurons. In the dcSC and vVM/VL very few neurons were GABA positive, while the EP was entirely devoid of GABA-immunoreactive neurons. These findings suggest that glutamatergic neurons are abundant in the entire subthalamic region. While glutamate, vGluT2 and EAAC1 immunohistochemistry were performed for the first time in an anuran, GABA-immunoreactivity and expression of glutamate decarboxylase (GAD, the GABA synthesizing enzyme) has already been determined in the

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brains of several anuran species. The absence of GABA-immunoreactive neurons in the EP of Bombina orientalis is in line with findings in Rana catesbeiana and Xenopus laevis (Hollis and Boyd, 2005), based on immunohistochemistry. In contrast, EP neurons in Xenopus laevis strongly express the gene xGAD-67, which encodes for glutamate decarboxylase (Brox et al., 2003), suggesting that EP neurons are at least capable of synthesizing GABA. The discrepancy between GABA immunoreactivity and xGAD-67 expression in Xenopus is puzzling, particularly as we do not know whether xGAD-67 labeling overestimates the number of GABAergic neurons, or GABA-immunohistochemistry underestimates it. Furthermore, it is also unknown if cytosolic GABA, which is synthesized by GAD-67 and connected to the tricarbolic acid cycle (Soghomonian and Martin, 1998; Waagepetersen et al., 1999), is correlated with synaptic GABA produced by GAD-65. Thus, with the methods available we can neither prove that GABAergic neurons (i.e. neurons releasing GABA from their synaptic terminals) exist in the subthalamic region, nor can we rule it out. However, since our GABA antibody labels numerous neurons in other brain areas, e.g. magnocellular nucleus of the torus semicircularis, posterior thalamic nucleus, or cerebellum (see supplementary figures), we believe that the most likely interpretation of our results is the presence of some GABAergic neurons in the dcSC and the vVM/VL, while they are rare or even absent in the EP. However, since GAD-67 mRNA has been demonstrated in Xenopus EP (see above), we cannot rule out species-specific differences in this respect. 4.1.2. Neurobiotin tracing We demonstrated projections from EP, dcSC, and ventral VM/VL to the SNr by retrograde tracing only, without control injections for anterograde tracing in our regions of interest. The reason for this was that especially the EP, but also the other regions are located near fiber tracts, and it would not be possible to inject tracer without labeling fibers of passage. Because direct and indirect pathway take the same course up to the level of the caudal diencephalon, only single cell labeling could disentangle them. 4.2. The subthalamic region in anuran amphibians The STN is located in prosomere 4 in reptiles, birds, and mammals (Puelles and Medina, 1994; Jiao et al., 2000). Unfortunately, prosomeres 4–6 are not well defined in anurans. The boundary between prosomere 3 and 4 (Puelles et al., 1996; Mila´n and Puelles, 2000) was initially thought to separate the ventral thalamus (prosomere 3) from more rostroventrally situated areas (prosomeres 4–6). In a more recent study (Brox et al., 2003), the ventral portion of the ventral thalamus is regarded to be part of prosomere 4. The boundary between prosomere 4 and 5 is not fully delineated as well, especially not in the basal plate where the subthalamic nucleus is to be found (Keyser, 1972; Altman and Bayer, 1978a,b). Therefore, the dcSC, EP, and/or vVM/VL may be derived from prosomere 4, since the lateral forebrain bundle is already located in prosomere 5 (Mila´n and Puelles, 2000). Hereafter, we will refer to these nuclei as the subthalamic region. 4.3. The anuran substantia nigra pars reticulata Although a number of brain areas project to the SNr, afferents from the STN constitute the main excitatory input (for review see Misgeld, 2004). To use efferents to the SNr as one criterion to identify the STN, we first have to delineate the location of the anuran SNr, which has been tentatively placed in the midbrain tegmentum (Marı´n et al., 1998a,b; Smeets et al., 2000). The SNr of mammals is defined by its GABAergic neurons and its input from

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the striatum (for review see Bolam et al., 2000). A prominent output is the nigrotectal pathway, which is mainly GABAergic (Chevalier et al., 1981). With GABA-immunoreactivity alone, we could not find the SNr, since GABA-ir cells are scattered throughout the tegmentum and do not form a discrete nucleus, neither in Bombina nor in other anuran species so far studied (Franzoni and Morino, 1989; Endepols et al., 2000; Simmons and Chapman, 2002). Instead, we defined the SNr by its terminal field of projections from the striatum (=end of direct pathway). In Bombina, the main part of the terminal field of striatotegmental projections lies in the lateral Av, and a second minor band of terminals is located at the boundary between Ad and Av. All other tegmental nuclei receive a considerable input from the striatum as well. A much clearer delineation of the striatotegmental terminal field could be achieved with substance P and enkephalin (substance P is the cotransmitter of GABAergic direct pathway striatal projections in all mammals, together with enkephalin in primates; Le´vesque et al., 2003) as well as GABAimmunohistochemistry. In Bombina, substance P, enkephalin, and GABA-immunohistochemistry resulted in identical staining and revealed two parts of a terminal fiber plexus, similar to our tracing studies: one is located among the cell bodies in the transition zone between dorsal and ventral tegmentum, the other in the neuropil lateral to the ventral tegmentum. In Rana esculenta, there is an enkephalinergic projection from the striatum to the midbrain tegmentum which follows the same pattern (La´za´r et al., 1990), and especially the band of axon terminals between dorsal and ventral tegmentum appears in all tracing studies with striatal injections (Rana temporaria: Vesselkin et al., 1980; Rana catesbeiana: Wilczynski and Northcutt, 1983; Rana perezi and Xenopus laevis: Marı´n et al., 1997b). It is unlikely that the whole area targeted by striatal axons is comparable to the SNr. The very rostral part (see Fig. 1G) overlaps with the rostral pedunculopontine tegmental nucleus defined by Marı´n et al. (1997c) due to its presence of cholinergic neurons, which continues caudally into the Ad. Av cells migrated into the lateral neuropil (where the ventrolateral part of the striatotegmental terminal field is located) have been termed nucleus profundus mesencephali (caudal part; cPrm; Ten Donkelaar, 1998; also called deep mesencephalic nucleus in mammals; Rodrı´guez et al., 2001) or superficial isthmal reticular nucleus (SIR; Nieuwenhuys and Opdam, 1976). The SIR/ cPrm seems not to be homologous to the SNr, because it neither contains GABAergic neurons nor does it project to the optic tectum (Marı´n and Gonza´lez, 1999; Sa´nchez-Camacho et al., 2002). We can therefore assume that the anuran SNr consists of two parts: a dorsal part in the Ad/Av transition area, where neurons receive direct striatal (enkephalinergic, substance P- and GABAergic) input to their somata and proximal dendrites, and a ventral part in the Av where striatal afferents mainly contact the distal dendrites. In the putative SNr areas only a few GABA-ir neurons can be found. At present, we cannot decide whether only those GABA-ir cells located within the striatal afferent plexus may be considered homologous to the mammalian SNr, or whether the dendrites of more rostrally or more caudally located GABA-ir neurons are also connected by striatal fibers and may therefore be part of the anuran SNr as well. Furthermore, whether one of those areas may be comparable to the substantia nigra pars lateralis (SNl; Kohno et al., 1984; Cebria´n et al., 2005) is also unknown. According to our definition of the SNr in Bombina (see Fig. 9) there is no close topographical relationship between SNr and SNc. However, the degree of overlap between the two structures is variable among vertebrates (for review see Marı´n et al., 1998a), and seems to depend on neuronal migration during development. In humans, dopaminergic SNc neurons originate in the floor plate of prosomeres 1–3, mesencephalon and isthmus. They migrate to their final positions, whereby they intermix with GABAergic SNr

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Fig. 9. Schematic drawing of areas associated with the indirect pathway in anurans. Colored dots represent neuronal cell bodies. (A) Glutamatergic neurons of the subthalamic region (blue). (B–D) Dopaminergic neurons of the SNc/VTA complex (purple), cholinergic neurons of the PPN (brown), neurons of the dorsal SNr (red; presumably cholinergic) and the ventral SNr (orange; some of them GABAergic, but transmitter mostly unknown), neurons of the SIR/cPrm (dark blue), and terminal fibers of the striatonigral pathway (green; immunoreactive for substance P, enkephalin, and GABA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

neurons of unknown origin (Verney et al., 2001). In amphibians (especially in salamanders, but also in frogs) neuronal migration is reduced due to increased genome size (Roth et al., 1994, 1997) which may explain the distance between SNc and SNr in anurans. It is not clear whether the SNr is the only structure in anurans where direct and indirect pathways meet. The other basal ganglia output nucleus, the entopeduncular nucleus (=globus pallidus internus in primates), does not exist separately from the globus pallidus externus in non-mammalian vertebrates (Reiner et al., 1998). The entopeduncular nuclei (anterior and posterior) of anuran amphibians received their name because of their close association with the lateral forebrain bundle, and are not necessarily homologous to the mammalian entopeduncular nucleus which was named after its location within the internal capsule. However, according to its chemoarchitecture, the anuran anterior entopeduncular nucleus may represent the caudal part of the globus pallidus (Marı´n et al., 1998c), and it is possible that internal and external pallidal neurons are intermixed in this area. Because pathway tracing is difficult in brain nuclei localized in/ around major fiber tracts, we focussed on the SNr rather than on the internal globus pallidus. Provided that both direct and indirect pathways end in the SNr, and that our classification of the anuran SNr is correct, analysis of subthalamo-nigral projections can help to identify the anuran STN. 4.4. Tracing of the indirect pathway To define the anuran STN we injected neurobiotin (separately) in the two parts of the SNr. For analysis we took only those specimens where many neurons in the rostral striatum (=striatum proper; Endepols et al., 2004) were retrogradely labeled, indicating that we had hit the terminal field of the direct pathway to the SNr. Assuming that the second part of the indirect pathway (the subthalamo-nigral projection) would target the same spot, we

looked for retrogradely labeled neurons in the subthalamic region. We found that the EP projected to the dorsal part of the SNr (the transition area between dorsal and ventral tegmentum) only, while the vVM/VL and dcSC projected to both dorsal and ventral SNr. Tracer injections into the globus pallidus (i.e., anterograde tracing of the first part of the indirect pathway) revealed that vVM/ VL, dcSC and EP were innervated by pallidal fibers. Because of its association with the lateral forebrain bundle the EP is in the best position to receive multiple pallidal inputs. This became most obvious in double fluorescence tracing experiments, where retrogradely labeled EP neurons (projecting to the SNr) were found to be embedded in anterogradely labeled fibers from the globus pallidus, while retrogradely labeled dcSC and vVM/VL neurons received less pallidal afferents. Furthermore, the EP is the nucleus with the most glutamate, vGluT2 and EAAC1 positive and the least GABA positive neurons, indicating the presence of numerous glutamatergic cells. While afferents and immunohistochemistry suggest that the EP may be comparable to the mammalian STN, the connections with the SNr rather support the vVM/VL and the dcSC as STN homologues. While only some EP neurons project to the SNr, one of its main targets is an area immediately caudal of the habenula, including the rostral part of the pretectum. However, this connection does not rule out the EP as an STN homologue, because in birds there is also a projection from the STN to the pretectum (Jiao et al., 2000). Neither the EP, nor the vVM/VL or the dcSC project back to the globus pallidus (Wilczynski and Northcutt, 1983; Marı´n et al., 1997a; this study), which is one of the main targets of the STN in both mammals and birds. If we regard this as an essential criterion, we must conclude that (1) an STN homologue does not exist in anuran amphibians. However, our results have shown that there is a polysynaptic pathway directed from the striatum via the globus pallidus to the putative SNr, which (2) may be considered as a homologue of an indirect pathway. To some

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Fig. 10. Diagram of the basal ganglia connections in anuran amphibians.

extent, these projections travel through the subthalamic area without an additional synapse (the pallido–nigral connection is shown in Fig. 7; see also Figs. 8 and 10), but relay neurons can also be found in the entire subthalamic region. A distinct STN homologue may be lacking, because (3) the subthalamic region may not have been parcellated in the sense of Ebbesson (1980), and may therefore share field homology with the amniote STN plus adjacent areas, e.g. zona incerta (for a critical review of the concept of field homology, see Puelles and Medina, 2002). The projection back to the globus pallidus may have developed later in evolution, possibly to subserve cross-talk between the increasingly complex basal ganglia-thalamocortical circuits (for review see Joel and Weiner, 1997). It is, however, still conceivable that (4) one area of the subthalamic region (EP, dcSC, or vVM/VL) may be the anuran homologue of the mammalian STN. Further studies with developmental marker genes can help to clarify this issue.

Acknowledgement Supported by Deutsche Forschungsgemeinschaft Wa 446/4, En 439/1.

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