Afferents of the lamprey striatum with special reference to the dopaminergic system A combined tracing and immunohistochemical studyкод для вставкиСкачать
THE JOURNAL OF COMPARATIVE NEUROLOGY 386:71–91 (1997) Afferents of the Lamprey Striatum With Special Reference to the Dopaminergic System: A Combined Tracing and Immunohistochemical Study MANUEL A. POMBAL,1,2 ABDELJABBAR EL MANIRA,1 AND STEN GRILLNER1* Nobel Institute of Neurophysiology, Department of Neuroscience, Karolinska Institutet, S-17177, Stockholm, Sweden 2Departamento de Biologı́a Fundamental, Facultad de Ciencias, Universidad de Vigo, E-36200, Vigo, Spain 1The ABSTRACT The origin of afferents to the striatum in lamprey (Lampetra fluviatilis) was studied by using fluorescein-coupled dextran-amines (FDA). Injection of FDA into the striatum retrogradely labeled several cell populations in the forebrain and the rostral rhombencephalon. No retrograde labeled cells were seen in the mesencephalon. A dopamine-specific antiserum was used to determined the distribution of dopaminergic perikarya and fibers. Many dopamine-immunoreactive (DA-ir) fibers were present throughout the brain, but the highest density of labeled fibers was in the mediobasal prosencephalon, especially in the striatum, the lateral hypothalamic area, and the neurohypophysis. Most DA-ir cells were located in the mediobasal diencephalon (preoptic region, nucleus commissurae postopticae, hypothalamus, and nucleus tuberculi posterioris). In the mesencephalon, only a few immunopositive cells were observed in the tectum opticum. In the rhombencephalon, DA-ir cells were observed in the isthmic region, dorsally to the descending trigeminal tract, and caudally to the posterior rhombencephalic reticular nucleus. The rostralmost spinal cord received many descending DA-ir fibers from the brainstem. Along the spinal cord, DA-ir neurons were also found, some of which projected to the medioventral surface, forming a prominent plexus. On the basis of double-labeling experiments, it is shown that the dopaminergic input to the striatum originates from the nucleus tuberculi posterioris. Thus, the striatum receives inputs from different structures, including a strong dopaminergic innervation from the diencephalon. Much of the dopaminergic system in Lampetra fluviatilis is basically similar to that seen in some teleosts, but it presents differences with other anamniote (elasmobranch) as well as amniote groups. J. Comp. Neurol. 386:71–91, 1997. r 1997 Wiley-Liss, Inc. Indexing terms: Lampetra fluviatilis; telencephalon; basal ganglia; fluorescent tracers; dopamine The vertebrate basal ganglia play an important role in the control of different types of motor behavior. Comparatively little is known about the organization of the basal ganglia in the lamprey, a vertebrate that separated from the main vertebrate evolutionary line 450 million years ago (Hotton, 1976). The lamprey nervous system is of interest not only from an evolutionary perspective but also because more knowledge is available concerning the neuronal circuitry in the brainstem-spinal cord than for most other vertebrates (for reviews, see Rovainen, 1979; Brodin and Grillner, 1990; Grillner et al., 1995). The striatum is one of the most prominent cell masses in the lamprey telencephalon medium (Johnston, 1912; Her- r 1997 WILEY-LISS, INC. rick and Oberchain, 1913; Heier, 1948). The striatum is composed of several layers of closely packed cells separated from the ependyma by a rather broad neuropil (Johnston, 1902; Heier, 1948). The telencephalon of lampreys consists of two well-developed, evaginated, dorsolat- Grant sponsor: Swedish Medical Research Council; Grant number: 3026, Grant number: 11562. *Correspondence to: Prof. Sten Grillner, The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden. E-mail: firstname.lastname@example.org Received 18 July 1996; Revised 17 April 1997; Accepted 22 April 1997 72 M.A. POMBAL ET AL. eral olfactory bulbs and cerebral hemispheres and an unevaginated telencephalon medium. Homologies between telencephalic cell groups in lampreys and other vertebrates are difficult to determine, but several pallial subdivisions have been proposed based on their topography and secondary olfactory afferents (Northcutt and Puzdrowski, 1988). Recent experimental studies on the connections of the lamprey telencephalon (Northcutt and Puzdrowski, 1988; Polenova and Vesselkin, 1993; Northcutt and Wicht, 1997) have focused on secondary and tertiary olfactory projections and major pallial connections. However, no studies have been performed on the connections of the subpallial structures. The limited information available concerning afferents and efferents of the lamprey striatum (Johnston, 1902; Heier, 1948) are based mainly on general and silver stainings. The striatum in lamprey has a chemical organization similar to that of other vertebrates, with neurons that are immunoreactive towards g-aminobutryic acid (GABA) and substance P and that are positive for acetylcho- line esterase (Pombal et al., 1995). In addition, it is also densely innervated by fibers that are immunoreactive to serotonin (5-HT; Brodin et al., 1988, 1990b; Pierre et al., 1992), substance P (Nozaki and Gorbman, 1986; Pombal et al., 1995), histamine (Brodin et al., 1990a), neurotensin (Brodin et al., 1990b), tyrosine hydroxylase (TH; Pierre et al., 1994), galanin (Jiménez et al., 1996), and enkephalin (Pombal et al., 1995). In mammals, the dorsal striatum is involved in sensorimotor and cognitive functions, whereas the ventral striatum is implicated in the processing of limbic information entering the basal ganglia (Alheid et al., 1990). A wide variety of sources of inputs to the striatum and nucleus accumbens have been described, with both the striatum and the nucleus accumbens receiving a very dense dopaminergic innervation (Parent, 1986; Smeets and Reiner, 1994b; Medina and Reiner, 1995), but the number of known efferent connections is more limited (Alheid et al., 1990). Abbreviations aon arn b c ca cc cgl ch cp cpi cpo cpr cpt cr cs ctp D dcf dh dhy dm dmc dn dp dth dV f go h hy igl III ip IV lc lco lpd lpv M M3 M5 mlf mn mp mra mrn mt nb nca ncp ncpi anterior octavomotor nucleus anterior rhombencephalic reticular nucleus commissura interbulbaris cerebellar plate or cerebellar commissura commissura anterior central canal of the spinal cord corpus geniculatum laterale chiasma opticum commissura posterior commissura postinfundibularis commissura postopticae commissura praeinfundibularis commissura posttectalis cell region of the spinal cord central stratum (tectum opticum) commissura tuberculi posterioris diencephalon dorsal column fibers dorsal horn of the spinal cord nucleus dorsalis hypothalami dorsomedial telencephalic neuropil dorsomedial column of the spinal cord dorsal nucleus of the octavolateral area dorsal pallium nucleus dorsalis thalami radix sensitivus nervi trigemini fasciculus retroflexus glomeruli olfactorii ganglion habenulae hypothalamus internal granular layer of the olfactory bulb nucleus nervi oculomotorii nucleus interpeduncularis nucleus nervi trochlearis lateral column of the spinal cord locus coeruleus dorsal part of the lateral pallium ventral part of the lateral pallium mesencephalon third Müller cell nucleus M5 of Schober medial longitudinal fasciculus medial nucleus of the octavolateral area medial pallium mesencephalic reticular area medial rhombencephalic reticular nucleus mesencephalic tegmentum nucleus of Bellonci nucleus commissurae anterior nucleus commissurae posterioris nucleus commissurae postinfundibularis ncpo ncpr nh nmlf noa np npo nrpo ntp nts ob os ot P p pch PP prn Ps ps R ra ri rpo rpos rpro s SC sd slf slh so ss st T to ts vc vd vhy VII vla vlp Vm vm vn vr vt vth X nucleus commissurae postopticae nucleus commissurae praeinfundibularis neurohypophysis nucleus fasciculi longitudinalis medialis nucleus olfactorius anterior nucleus praetectalis nucleus praeopticus nucleus recessus postopticus nucleus tuberculi posterioris nucleus tractus solitarii olfactory bulb optic stratum (tectum opticum) tectum opticum pineal organ ventromedial plexus of the spinal cord plexus choroideus parapineal organ posterior rhombencephalic reticular nucleus pineal stalk periventricular stratum (tectum opticum) rhombencephalom raphe region recessus infundibuli recessus postopticus recessus posterior recessus praeopticus nucleus septi spinal cord saccus dorsalis spinal lemniscal fibers sulcus limitans of His subcommisural organ superficial stratum (tectum opticum) striatum telencephalon tractus opticus torus semicircularis ventral column of the spinal cord ventriculus diencephali nucleus ventralis hypothalami nucleus motorius nervi facialis ventriculus lateralis anterior telencephali ventriculus lateralis posterior telencephali nucleus motorius nervi trigemini ventriculus mesencephali ventral nucleus of the octavolateral area ventriculus rhombencephali ventriculus medius telencephali nucleus ventralis thalami nucleus motorius nervi vagi STRIATAL AFFERENTS IN LAMPREY Dopamine (DA), together with adrenaline and noradrenaline, are the major catecholamines used by the nervous system, with TH as the key enzyme involved in their biosynthesis. Current understanding of catecholaminergic systems in the lamprey forebrain is based mainly on studies of the distribution of monoamine oxidase (MAO; Tsuneki et al., 1975) and formaldehyde-induced histofluorescence (FIF; Honma, 1969; Honma and Honma, 1970; Baumgarten, 1972; Konstantinova, 1973; Ochi and Hosoya, 1974; Tsuneki et al., 1975) and on immunohistochemical markers, such as noradrenaline (Steinbusch et al., 1981b), TH (Brodin et al., 1990a; Pierre et al., 1994), and aromatic amino acid decarboxylase (AADC; Mahouche et al., 1994). Most of these studies have focused on the hypothalamohypophyseal system. Catecholaminergic terminals in the striatum were initially described in the river lamprey by Baumgarten (1972), and, more recently, immunohistochemical studies have revealed the presence of TH-immunoreactive (ir) fibers (Pierre et al., 1994). Since the development of antibodies against DA (Geffard et al., 1984), many studies have been carried out in different groups of vertebrates (for review, see Smeets and Reiner, 1994a). Some preliminary results with DA antibodies were also reported in larval (Yáñez et al., 1992) and adult (Pierre et al., 1994) lampreys. In lampreys, a small catecholaminergic cell group suggested to be a possible homologue of the substantia nigra in amniotes has been described in the region of the posterior tubercule (Baumgarten, 1972), which is incorporated in the base of the midbrain in tetrapods. In the spinal cord, two populations of DA-ir cells have recently been described, cerebrospinal fluid (CSF)-contacting cells around the central canal and 5-HT/DA-containing cells, which form a ventromedial plexus (Schotland et al., 1995, 1996). The aim of the present study was threefold: 1) to describe the input to the striatum from different forebrain structures by injection of the tracer fluorescein-coupled (FDA) and biotinylated (BDA) dextran-amines, 2) to reveal the distribution of dopaminergic cells and fibers in the lamprey forebrain and brainstem by using antibodies against DA, and 3) to determine the cells of origin of the dopaminergic innervation of the lamprey striatum by using double-labeling experiments with BDA and a TH antiserum. Part of these results have been published in abstract form (Pombal et al., 1995, 1996). MATERIALS AND METHODS Animals This study was performed on 38 adult (20–35 cm in total length) river lampreys (Lampetra fluviatilis L.) that were captured in Söderhamn, Sweden and kept in aerated freshwater aquaria at 4–10°C. Fourteen animals were used for fluorescence-tracing studies, 16 for immunocytochemistry, and eight for double labeling. Prior to experimentation, the animals were anesthetized with 0.01% solution of tricaine methanesulphonate (MS-222; Sandoz, Basel, Switzerland) in fresh water. The protocols used in this study were approved by the ethical committee of animal experiments (Stockholm, Sweden). In vitro fluorescence-tracing experiments Animals were decapitated, and the brains were dissected out in a cooled physiological Ringer’s solution with the following composition (in mM): 138 NaCl, 2.1 KCl, 1.8 73 CaCl2, 1.2 MgCl2, 4 glucose, 0.5 L-glutamine and 2 Hepes. The solution was bubbled with 100% O2 for 15 minutes, and the pH was adjusted to 7.4 with NaOH. The pineal and parapineal organs and the plexus choroideus were removed. The commissura interbulbar and, sometimes, the commissurae habenular and posterior were cut to facilitate access to the impar telencephalic ventricle. FDA (3,000 MW; Molecular Probes, Eugene, OR) paste (a concentrated solution of the tracer in distilled water that was desiccated on the tip of a 000 insect pin) was applied to the brains at the level of the striatum region for 1–2 minutes. The brains were subsequently washed to remove the excess tracer and kept at 4°C in the same Ringer’s solution for 24–65 hours to allow transport of the FDA. The brains were then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 24–96 hours, washed in PB, and treated for 30 minutes with 1% sodium borohydride in the same buffer. After washing with 0.1 M PB, pH 7.4, and cryoprotection with 15% and 30% sucrose solutions in the same buffer, the brains were mounted in OCT (Optimal Cutting Temperature; Miles Inc., Elkhart, IN), frozen with CO2, and transversely sectioned at 30 µm by using a Leitz 1720 digital cryostat. Sections were then collected and mounted on gelatin-coated slides and were examined and photographed with a Nikon microphot-FXA microscope. Immunocytochemical procedure Animals were decapitated, and the brains were quickly removed in physiological Ringer’s solution and fixed overnight at 4°C in 4% glutaraldehyde (GTA) in 0.1 M PB, pH 7.4. The brains were subsequently washed in PB (3 3 10 minutes) and treated for 30 minutes with 1% sodium borohydride to reduce Schiff bases caused by GTA fixation. Transversal, sagittal, or horizontal cryostat sections (16 µm thick) were obtained after cryoprotection with 30% sucrose in 0.1 M PB. A specific rabbit DA antiserum (Steinbusch et al., 1991) and three different immunostaining protocols (peroxidase-antiperoxidase: PAP, n 5 10; streptavidin-biotin: SAB complex, n 5 4, and fluorescence, n 5 2) were used. For the PAP or SAB complex, all sections were treated for 20–30 minutes with 0.3% H2O2 to abolish pseudoperoxidase activity in erythrocytes. Sections were washed in 0.1 M phosphate-buffered saline (PBS; pH 7.4) and subsequently incubated in a humid chamber. For the SAB complex protocol, sections were incubated at 4°C overnight with the DA antiserum raised in rabbit (Steinbusch et al., 1991; dilution: 1:500 to 1:1,000), rinsed in PBS (3 3 10 minutes), incubated for 30 minutes at room temperature with biotinylated goat anti-rabbit IgG (Dakopatts, Glostrup, Denmark; diluted 1:200), rinsed with PBS, and incubated again with StreptABComplex/horseradish peroxidase (HRP; Elite, ABC kit; Vector Laboratories, Burlingame, CA) for 30 minutes. Following extensive rinsing, the complex was developed by incubation in 3,38-diaminobenzidine (Sigma, Stockholm, Sweden; 50 mg/100 ml) and 0.003–0.01% hydrogen peroxide for 8–10 minutes. For the PAP protocol, sections were incubated sequentially by using the same DA antiserum (dilution 1:800 to 1:1200) at 4°C overnight, goat anti-rabbit IgG (Dakopatts; diluted 1:60) for 1 hour at room temperature, and rabbit PAPcomplex (Dakopatts; diluted 1:300) for 45 minutes; the complex was developed as described above. Sections were then rinsed thoroughly, dehydrated in graded alcohols, and coverslipped. 74 M.A. POMBAL ET AL. In experiments conducted by using the indirect immunofluorescence method, the sections were incubated with rabbit DA antiserum (Steinbusch et al., 1991; dilution 1:400 to 1:600) for 18–24 hours at 4°C, rinsed in PBS, and then incubated for 1 hour at room temperature with fluorescein isothiocyanate (FITC)-coupled goat anti-rabbit antibodies (Boehringer-Mannheim, Germany; dilution 1:40 to 1:80). After being rinsed in PBS, the sections were mounted in a glycerol-PBS mixture (3:1). All sections were examined and photographed in a Nikon microphot-FXA microscope. The different immunostaining procedures (PAP, SAB complex, and fluorescence) produced the same pattern of DA immunoreactivity, although some cell populations showed stronger labeling when using the SAB complex than when using immunofluorescence or PAP staining. Double-labeling experiments For double-labeling experiments, we used the same procedure as described by Marı́n et al. (1995). Briefly, BDA (3,000 MW; Molecular Probes Europe, Amsterdam, Netherlands) was applied in the striatum (as described above for FDA). After survival times of 60–65 hours, the brains were fixed overnight in 4% paraformaldehyde in 0.1 M PB, pH 7.4, cryoprotected with 30% sucrose, and transversally, horizontally, or sagitally sectioned at 16 µm. Sections were then incubated for 48 hours at 4°C with a rabbit TH antiserum (Markey et al., 1980; dilution 1:400 to 1:600). They were subsequently incubated with an FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab., Inc., West Grove, PA; dilution 1:60) and a Texas-redconjugated streptavidin complex (Vector Laboratories; dilution 1:100) to visualize TH and BDA, respectively. The sections were mounted with a glycerin-PB medium, coverslipped, examined, and photographed with a Nikon microphot-FXA microscope. In some cases (four brains), BDA was visualized by using a StreptABComplex/HRP (Elite, ABC kit; Vector Laboratories) for 60 minutes and was subsequently developed by incubation in 3,38-diaminobenzidine (Sigma; 50 mg/100 ml) and 0.003–0.01% hydrogen peroxide for 8–10 minutes. Nomenclature For the forebrain and the mesencephalic areas, we have used the anatomical nomenclature of Heier (1948) and Schober (1964) and, for the pallial subdivisions, that of Northcutt and Puzdrowski (1988). For the rhombencephalon, we have followed Nieuwenhuys (1977), Koyama et al. (1989), and Ronan and Northcutt (1990). The subdivisions of the spinal cord are modified from Van Dongen et al. (1985). RESULTS The distribution of labeled cell bodies revealed by injections of FDA or BDA into the striatum will be described first. It is demonstrated that the striatum receives inputs from most telencephalic nuclei (cf. Fig. 1), including all major divisions of the pallium. The remaining afferents have a diencephalic or rhombencephalic origin and include different thalamic and hypothalamic nuclei, the nucleus tuberculi posterioris, the presumed locus coeruleus, and the raphe region. A detailed description of the dopamine immunoreactivity in the whole lamprey brain and spinal cord will then be described. A very dense dopaminergic innervation of the lamprey striatum is demonstrated as well as the presence of a well-developed dopaminergic system. Afferent projections to the striatum To characterize the cell populations projecting to the striatum, FDA or BDA was injected into this nucleus (see Materials and Methods). The locations of retrogradely labeled fibers and cells after these injections are shown schematically in transversel sections from different levels (Fig. 1) and are summarized in a midsagittal projection (Fig. 2). All striatal injections (st; see Fig. 1B) included both the periventricular neuropil and the layer of cell bodies underneath. After these injections, numerous labeled fibers were observed in the dorsal, lateral, and ventral areas adjacent to the injection site. Retrogradely labeled neurons occurred immediately rostral in the nucleus commissurae anterior (nca; Figs. 1A, 2) and the nucleus septi (s; Figs. 1A, 2) and, ventrally, in the nucleus praeopticus (npo; Figs. 1B, 2). Some fluorescent cells were also present in the dorsal pallium (dp; Figs. 1B,C, 2) and in the ventral part of the medial pallium (mp; Figs. 1C, 2). Larger numbers of retrogradely labeled cells were seen both laterally in the internal granular layer of the olfactory bulb (igl; Figs. 1A,B, 2) and caudolaterally in the ventral part of the lateral pallium (lpv; Figs. 1B,C, 2, 3A). A few cells were also retrogradely labeled in the dorsal part of the lateral pallium (lpd; Figs. 1B,C, 2). Caudal to the striatum, labeled fibers coursed in the ventral and ventrolateral directions. Labeled fibers running ventrally divided into a medial and a ventral bundle (Fig. 1C). The medial bundle decussated in the commissura postopticae (cpo; Fig. 1C) and turned either dorsally toward the contralateral nucleus ventralis thalami (vth) or ventrally toward the hypothalamus (dhy and vhy), where some retrogradely labeled cells were seen (Figs. 1D,E, 2). The ventral bundle reached the nucleus commissurae postopticae (ncpo; Fig. 1C), the nucleus recessus postopticus (nrpo; Fig. 1C), and the nucleus commissurae praeinfundibularis (ncpr; Fig. 1F). Most of the remaining labeled fibers reached the caudal hypothalamus, the nucleus ventralis thalami, and the caudalmost diencephalon (Figs. 1D–F, 3E). Numerous cells were retrogradely labeled in the nucleus dorsalis thalami in a subhabenular position (dth; Figs. 1E, 2, 3B), the nucleus ventralis thalami (vth; Figs. 1D,E, 2, 3B,C), and the nucleus dorsalis and ventralis hypothalami (dhy and vhy; Figs. 1D,E, 2, 3D,E). Ipsi- and contralaterally labeled cells in the nucleus ventralis thalami were located in both a subependymal and a laterally displaced area. The subependymal cells were small, elongated in shape, and located perpendicular to the ventricular surface, whereas the laterally located cells were bigger and polygonal (Fig. 3C). Labeled cells in the hypothalamic areas were of both CSF- and non-CSFcontacting types (Fig. 3D,E). Some retrogradely labeled cells also occurred within the nucleus tuberculi posterioris (ntp; Figs. 1F, 2, 3D; cf. also Fig. 9B) in the basal diencephalon. These cells were small and were either round or pear-shaped. A few of them were located in the midline or slightly contralateral to the injection site. Some retrogradely labeled cells were also present in the rostral rhombencephalon in both the isthmus region, constituting the presumed locus coeruleus (lc; Fig. 1G; cf. also Fig. 9D), and another area close to the midline below the trigeminal Fig. 1. A–H: Diagrammatic representation of the distribution of retrogradely labeled cell bodies (large dots) and fibers (dashed lines and small dots) in transverse sections of the river lamprey brain following a unilateral injection of fluorescein-coupled dextran-amines (FDA) into the left striatum. The black oval region in B indicates the site of the tracer injection, and the shaded area shows the subsequent extent of its diffusion. On the left of A–H, short dashed lines represent fiber tracts, and long dashed lines delineate the different cell populations. The sections are arranged from rostral to caudal, and the level of each section is shown in a sagittal view of the brain in Figure 2. For abbreviations, see list. Scale bar 5 1 mm. 76 M.A. POMBAL ET AL. In the telencephalon proper, the distribution of DA-ir fibers was most pronounced in the basal portion, particularly in the periventricular neuropil of the striatum (Figs. 4C,D, 5B, 6C,D). Numerous DA-ir fibers were also present in the nucleus septi, the nucleus olfactorius anterior, the nucleus commissurae anterior (Fig. 4B), and the ventral part of the lateral pallium (Fig. 4C,D). In contrast, only a few labeled fibers were seen in the dorsal part of the lateral pallium (Fig. 4D), medial pallium (Fig. 4C–E), and dorsal pallium (Fig. 4C,D). Diencephalon Fig. 2. Sagittal view of the river lamprey brain showing the summed distribution of labeled cell bodies (dots) following a unilateral injection of FDA into the striatum (st). Levels A–H illustrate the relative positions of the transverse sections shown in Figure 1. For abbreviations, see list. Scale bar 5 1 mm. motor nucleus, forming part of the raphe region (Figs. 1H, 3F). DA immunocytochemistry In all vertebrates studied thus far, the striatum and the nucleus accumbens are densely innervated by DA-ir fibers originating mostly from the substantia nigra and the ventral tegmental area (see Parent, 1986; Smeets and Reiner, 1994b; Medina and Reiner, 1995). By using a specific DA antibody, several groups of DA-ir neurons were found in the lamprey (cf. Figs. 4, 5). The most prominent populations were located in the diencephalon, whereas fewer DA-ir populations were located in the telencephalon, mesencephalon, and rhombencephalon. The major anatomical localization of DA-ir perikarya and fibers in L. fluviatilis is illustrated schematically in selected transverse sections through the brain and spinal cord (Fig. 4) and projected onto a midsagittal plane (Fig. 5). A detailed description of the dopaminergic system is reported below for each of the major brain subdivisions (i.e., telencephalon, diencephalon, mesencephalon, and rhombencephalon) and also in the spinal cord. Telencephalon Perikarya. The internal granular layer of the olfactory bulb contained numerous scattered and weakly labeled dopaminergic cell bodies (igl; Figs. 4A–C, 5, 6A). These cells were small (5–12 µm in diameter) and round or ovoid shaped. Round or elongated DA-ir cell bodies were also found in the nucleus commissurae anterior (nca; Figs. 4B, 5, 6B). Some of these dopaminergic cell bodies had a short apical process that reached and contacted the recessus praeopticus (rpro). Fibers. Some short labeled fibers were found in the olfactory bulb that originated from dopaminergic cells of the internal granular layer (igl; Figs. 4A–C, 6A). The dorsomedial telencephalic neuropil was also innervated by DA-ir varicosities, presumably originating from the same DA-ir neurons (Figs. 4C,D, 6A). Perikarya. The medioventral part of the diencephalon contained most of the DA-ir cell bodies in the brain of adult L. fluviatilis (Figs. 4C–G, 5A, 7). The rostralmost DA-ir neurons were located around the chiasma opticum, in the region of the nucleus praeopticus (npo; Figs. 4C, 5A, 6B,C,E). This nucleus contained immunoreactive fusiform cell bodies (approximately 10 3 25 µm in size) arranged perpendicularly to the optic fibers. Most of these DA-ir cells were located slightly dorsal to the optic fibers and possessed short CSF-contacting processes that reached and protruded into the recessus praeopticus, whereas round and dorsally located cells had no CSF-contacting processes. Caudal to this region, some DA-ir cells in the nucleus commissurae postopticae (ncpo; Figs. 4D, 5A, 6B,E) formed a sparse population of small (10–15 µm), round or slightly fusiform cells near the ventricular surface. The most conspicuous population of DA-ir cell bodies was found in the caudal hypothalamic walls of the diencephalic ventricle. The nucleus dorsalis and ventralis hypothalami (dhy and vhy; Figs. 4E,F, 5A, 6E, 7B,C) as well as the nucleus commissurae postinfundibularis (ncpi; Figs. 4G, 5A, 7C–E) contained numerous DA-ir cell bodies arranged in one to three rows beneath and among the ependymal cells, parallel to the recessus infundibuli and posterior. These cells were mostly bipolar, but some pearshaped perikarya were also seen in the ependymal layer. Most, if not all, of these cells had apical CSF-contacting processes that reached and protruded into the diencephalic (third) ventricle and long basal processes that coursed to the ventrolateral neuropil, forming a dense immunoreactive plexus (Figs. 4E,F, 6A,B). Some weak DA-ir cells were also located in the nucleus tuberculi Fig. 3. Photomicrographs of several transverse sections through the brain of the river lamprey illustrating retrogradely labeled cell bodies and fibers following biotinylated dextran amine (BDA; A,B,F) or FDA (C–E) injection into the left striatum. A: Retrogradely labeled cells (arrowheads) and fibers in a horizontal section through the ventral part of the lateral pallium following an ipsilateral injection. The star indicates the ventriculus lateralis posterior telencephali. B: Labeled cell bodies (arrowheads) in the ipsilateral nucleus dorsalis thalami. The asterisk indicates the fasciculus retroflexus. The arrow indicates a labeled cell in the ventral thalamus. C: Retrogradely labeled cell bodies in the contralateral nucleus ventralis thalami in both periventricular (arrows) and laterally displaced (arrowhead) positions. D: Labeled cells in the nucleus tuberculi posterioris (arrow) and the nucleus ventralis hypothalami (arrowheads). Note that only one labeled cell is located on the contralateral side of the nucleus ventralis hypothalami (arrowhead on the right side). E: Labeled cells of both cerebrospinal fluid- (CSF; arrows) and non-CSF-contacting (arrowheads) types in the nucleus dorsalis and ventralis hypothalami. F: Retrogradely labeled cells in the raphe region (arrows) following BDA injection into the striatum. For abbreviations, see list. Scale bars 5 200 µm in A,B,D,E, 100 µm in C,F. STRIATAL AFFERENTS IN LAMPREY 77 Figure 3 78 Fig. 4. A–N: Schematic drawings of representative rostrocaudal transverse sections showing the location of dopamine-immunoreactive (DA-ir) perikarya (large dots, non-CSF-contacting cells; triangles, CSF-contacting cells) and fibers (dashed lines and small dots). The M.A. POMBAL ET AL. sections are arranged from rostral to caudal, and the level of each section is shown in the sagittal view of the brain in Figure 5A. For abbreviations, see list. Scale bar 5 1 mm. STRIATAL AFFERENTS IN LAMPREY 79 Figure 4 posterioris (ntp; Figs. 4G, 7F). These small (5–10 µm), round cells were distributed around the midline, dorsal to the recessus posterior. In another species of lamprey, Petromyzon marinus, cells in this nucleus showed a stronger DA immunoreactivity and were somewhat larger and more numerous (data not shown). This region has been considered by Baumgarten (1972) as the cytoarchitectonic area equivalent to the ventral tegmental area-substantia nigra of other vertebrates. Fibers. Dopaminergic cells in the preoptic region gave rise to a diffuse fiber tract that coursed caudally towards the hypothalamus, joining the tractus olfactohypothalamicus of Heier (1948), which passes by in close proximity (Fig. 5B). This DA-ir fiber tract may innervate the neurohypophysis and, if so, form a well-developed neurosecretory pathway (see also Nieuwenhuys, 1977). Some other fine efferent fibers from the preoptic region followed and joined the tractus opticus, also coursing caudally to thalamic levels (Heier, 1948). The mediobasal diencephalon displayed a very dense plexus of DA-ir fibers, originating from CSF-contacting cells of the dhy and the vhy (Figs. 5B, 7). After reaching the lateral part of the brain, these immunopositive fibers gave rise to two main fiber bundles. One coursed rostrally to the telencephalon and innervated mainly the striatum region, whereas the other projected caudally to the mesencephalon, the rhombencephalon, and even the dorsal column of the rostralmost region of the spinal cord (Figs. 5B, 7C). (Continued.) Some labeled fibers also turned dorsally to innervate the corpus geniculatum laterale (cgl; Fig. 4F) and the pretectal region (Fig. 4G). A few DA-ir fibers were also seen crossing in the commissura posterior (cp; Fig. 4G), continuing to the contralateral pretectal region. In the epithalamus, the habenula was almost completely devoid of DA-ir fibers (Figs. 4E, 5B). DA-ir fibers were absent from the pineal and parapineal organs (Figs. 4B,C, 5B). The neural lobe of the hypophysis showed abundant DA-ir fibers, mainly in its ventrocaudal aspect (Figs. 4D,E, 5B, 7C). Mesencephalon Perikarya. In the mesencephalon, only a few small (5 µm in diameter), round, weakly labeled DA-ir cells were observed in the caudal part of the superficial stratum of the tectum opticum (Figs. 4H, 5A, 8A). These cells were monopolar, and their short basal processes coursed deeply into the tectum opticum. No DA-ir cell bodies were detected in the ventral mesencephalon (Fig. 4H). Fibers. The lamprey mesencephalon received a considerable dopaminergic innervation from the diencephalon. Dorsally, the periventricular and central strata of the tectum opticum received numerous DA-ir fibers (Figs. 4H, 5B, 8A), whereas the optic and superficial strata were devoid of immunoreactive fibers. A moderate dopaminergic innervation was present in the torus semicircularis, retinopetal nuclei (nucleus M5 of Schober and mesencephalic 80 M.A. POMBAL ET AL. Fig. 5. Diagrams summarizing the distribution of DA-ir cells and the main fiber tracts in the adult lamprey brain and spinal cord, as projected in a sagittal view. A: Schematic drawing illustrating the localization of DA-ir perikarya (dots). Levels A–N correspond to the transverse sections shown in Figure 4. B: Main dopaminergic fiber tracts (dashed lines) in a midsagittal view of the lamprey brain. Note that the most prominent tracts, originating from the diencephalon, course rostrally to the basal telencephalon and caudally to the brainstem. Dots represent DA-ir perikarya as in A. For abbreviations, see list. Scale bar 5 1 mm. reticular area) and mesencephalic tegmentum (Fig. 4H). Most of the caudally projecting dopaminergic fibers in the mesencephalon were located in the region between the oculomotor nucleus and the habenulointerpeduncular tract, which, in itself, was devoid of immunopositive fibers along its whole extent (Figs. 4G–I, 7F). STRIATAL AFFERENTS IN LAMPREY Rhombencephalon Perikarya. Some weakly stained DA-ir cell bodies were observed in the ventromedial part of the isthmic nuclei (anterior rhombencephalic reticular nucleus or nucleus isthmi rhombencephali ventralis medialis of Pierre et al., 1992; Figs. 4I, 5A, 8B). These cells were round or pear-shaped and had their basal processes directed toward the lateral neuropil. In the rhombencephalon proper, a few DA-ir cells were seen in the ventral nucleus of the octavolateral area, just dorsal to the most rostral part of the descending trigeminal tract (Figs. 4J, 5A, 8C,D). These cells were small (5–8 µm) and round or ovoid in shape. Caudally, at the level of the posterior rhombencephalic reticular nucleus, a row of small DA-ir cells was located around the midline (Figs. 4L, 5A, 8E; CSF-contacting type). Some DA-ir cells were also located dorsally near the sulcus limitans of His in the presumed nucleus tractus solitarii (Figs. 4L, 5A, 8E). Fibers. The rhombencephalon was heterogeneously innervated with DA-ir fibers, which were widely distributed along its rostrocaudal extent. In the isthmic region, numerous DA-ir fibers were seen in the whole tegmentum and in the so-called cerebellar commissura (Figs. 4I, 8B). In the octavolateral area, dopaminergic fibers were abundant and clearly delineated the dorsal and medial nuclei (Figs. 4J,K, 8C), where electro- and mechanoreceptive fibers of the lateral line system are distributed (Ronan and Northcutt, 1987). In addition, the ventral nucleus (Figs. 4J–L, 8C,D) of this area exhibited a dense and homogeneous innervation by DA-ir fibers. Most DA-ir fibers, however, were distributed in the ventromedial tegmentum of the basal plate. Some fibers also formed a diffuse periventricular neuropil that bordered the motor nuclei (V–XII; Figs. 4J–L, 8C–E) of the visceral column. In the caudal rhombencephalon, most DA-ir fibers shifted dorsally to enter the dorsal column of the spinal cord (Figs. 4L,M, 8E,F). 81 Catecholaminergic innervation of the lamprey striatum BDA tracing was combined with indirect immunofluorescence for TH (n 5 8; see Materials and Methods) to study the origin of the catecholaminergic innervation of the lamprey striatum. The immunohistochemical results with a polyclonal TH antibody were largely similar to those reported by Pierre et al. (1994). Thus, we will describe only briefly certain differences in order to compare TH- and DA-ir structures (see also Table 1). In our experiments, no TH-ir cells were found in the nucleus olfactorius anterior, nucleus dorsalis thalami, nucleus commissurae posterioris, or nucleus tractus solitarii. Two different TH-ir cell populations were present in the region of the posterior tuberculum, i.e., the nucleus tuberculi posterioris, formed by small, round, non-CSF-contacting cells located close to the midline and the nucleus paratubercularis posterior, which contains larger and lateroventrally displaced CSFcontacting cells. Following BDA injections into the striatum combined with TH immunocytochemistry, double-labeled cells were found in both the nucleus tuberculi posterioris (Fig. 9A,B) and the presumed locus coeruleus (Fig. 9C,D). In the nucleus tuberculi posterioris, TH-ir cells abundant, with strong immunoreactivity, and were somewhat clustered in a small region (Fig. 9A). In the presumed locus coeruleus, TH-ir cells were relatively few in number, with weak immunoreactivity, and were scattered dorsoventrally in the lateral part of the gray (Fig. 9C). The maximum number of double-labeled cells was 7 of 16 retrogradely labeled cells in the nucleus tuberculi posterioris and 5 of 8 retrogradely labeled cells in the locus coeruleus. DISCUSSION Spinal cord The results of this study establish the presence of a dense dopaminergic innervation of the neuropil of the striatum in the lamprey. The origin of this innervation was determined by combining TH immunohistochemistry with tracing techniques (BDA) by means of double fluorescence. Perikarya. Similar to the very caudal part of the rhombencephalon, DA-ir cell bodies were found ventral to the central canal along the whole extent of the spinal cord (Figs. 4M,N, 5A, 8F). These cells were small (10 3 6 µm) and bipolar and had a short apical process that reached the central canal and terminated as a bulb in close apposition to the Reissner fiber. Some non-CSF-contacting dopaminergic cells were also located ventral or slightly lateral to the central canal (Figs. 4M,N, 8F). The detailed morphology and synaptic connectivity of the intraspinal dopaminergic system has been published previously (see Schotland et al., 1996). Fibers. Many descending DA-ir fibers from the brainstem were found in the dorsal part of the rostralmost spinal cord (first segments; Figs. 4M, 8F). More caudally, the number of these fibers decreased rapidly, and only a few could be followed to the trunk region. Some DA-ir fibers originated from CSF-contacting cells and formed a small dopaminergic plexus dorsal to the central canal, whereas other coursed to the lateral gray matter (Schotland et al., 1996). Ventrally directed processes from intrinsic non-CSF-contacting dopaminergic cells gave rise to a prominent fiber plexus in the ventromedial column of the spinal cord (Figs. 4M,N, 8F; Schotland et al., 1996). Injections into the striatum region were performed through the telencephalic ventricle but not from the external part of the brain, in order to avoid or minimize the labeling of ascending and descending fibers that course in the lateral forebrain bundle and tractus opticus. In some experiments, however, parts of these fiber tracts were also labeled. After injections into the striatum, numerous cell populations from a range of telencephalic, diencephalic, and rhombencephalic sources were identified, some of which have been reported previously (Polenova and Vesselkin, 1993; Northcutt and Wicht, 1997; see below). The numbers of afferent striatal cells in the nucleus tuberculi posterioris that project to the striatum were comparatively low, even after a long transport time. In some experiments with FDA, no labeled cells were obtained in the caudal diencephalon (dorsally in the nucleus dorsalis thalami and ventrally in the nucleus tuberculi posterioris). In immunohistochemical experiments, several cells were stained for DA in the nucleus tuberculi posterioris; thus, it could be anticipated that this nucleus has more prominent projections to striatum. Retrogradely labeled cells in the pre- Technical comments 82 M.A. POMBAL ET AL. Figure 6 STRIATAL AFFERENTS IN LAMPREY sumed locus coeruleus and the raphe region were obtained only when BDA was used as a tracer. Although similar survival times allow in vitro labeling of the striatal afferents, no labeled cells were seen in the nucleus of the solitary tract of the lamprey. The low number of retrogradely labeled cells in some striatal sources and the absence of labeled cells in the nucleus of the solitary tract could be explained by the long transport distance between these structures and the striatum and the small diameter of the retrogradely labeled fibers. The limitations of the technique for retrograde labeling, thus, should be taken into account when interpreting negative results. Striatal afferents from the dorsal thalamus Based on the number of retrogradely labeled cells, the major input to the striatum was the nucleus dorsalis thalami. In stained histological material, fibers apparently originating from the striatal area were traced to the nucleus dorsalis thalami by Johnston (1902) and Tretjakoff (1909), forming the tractus thalamofrontalis. Injections of DiI in the dorsal thalamus (Ichthyomyzon unicuspis; Northcutt and Wicht, 1997) also revealed the presence of varicose fibers in the neuropil lateral to the ipsilateral striatum. However, such a projection was not revealed by injection of HRP and HRP-wheat germ agglutinin (WGA) (L. fluviatilis; Polenova and Vesselkin, 1993). Projections from the nucleus dorsalis thalami to the striatum have been shown in amphibians (Wilczynski and Northcutt, 1983; Wicht and Himstedt, 1988; Dubé et al., 1990; González et al., 1994c; Marı́n et al., 1997a) and reptiles (Parent, 1976; Lohman and van Woerden-Verkley, 1978; González et al., 1990). The thalamic intralaminar nucleus in mammals sends a prominent projection to the striatum, whereas a minor input is provided by the lateral posterior/pulvinar complex (see Butler, 1994). The lamprey nucleus dorsalis thalami receives direct connections from the contralateral retina (Vesselkin et al., 1980; De Miguel et al., 1990), all major divisions of the pallium, the nucleus praeopticus, hypothalamus, optic Fig. 6. DA-ir cells and fibers in the forebrain of the river lamprey. A: Sagittal section through the evaginated part of the lamprey telencephalon showing DA-ir cells in the internal granular layer (igl) of the olfactory bulb. Note also the presence of immunoreactive terminals in the dorsomedial telencephalic neuropil (dm) as well as immunoreactive fibers in the nonolfactory part of the telencephalon. The dashed line shows the limit between the olfactory (right) and nonolfactory (left) parts of the telencephalon. B: Sagittal section illustrating DA-ir cell bodies in the nucleus commissurae anterior (nca), nucleus praeopticus (npo), and nucleus commissurae postopticae (ncpo). The arrow points to a displaced cell of the nucleus praeopticus. The star indicates the recessus praeopticus. C: Transverse section showing DA-ir cells in the nucleus praeopticus (npo) and DA-ir fibers in the basal telencephalon, which are particularly abundant in the periventricular neuropil that borders the striatum (st). Note that some of the non-CSF-contacting DA-ir cells are dorsally displaced (arrows). D: Horizontal section showing the dopaminergic innervation of the basal telencephalon. A conspicuous dopaminergic plexus is located medial to the striatum (st), whereas only a few fibers course to the lateral evaginated part (star). E: Horizontal section showing CSF-contacting cells in the nucleus praeopticus (npo) and nucleus dorsalis hypothalami (dhy) and non-CSF-contacting cells in the nucleus commissurae postopticae (ncpo). The star indicates the recessus praeopticus. A dense plexus of dopaminergic fibers is seen in the basal plate, whereas the tractus opticus (to) is devoid of immunoreactive fibers. For other abbreviations, see list. Scale bars 5 200 µm in A–C,E, 400 µm in D. 83 tectum, midbrain tegmentum, and dorsal isthmal gray as well as secondary and tertiary olfactory projections (Polenova and Vesselkin, 1993; Northcutt and Wicht, 1997). It has also been suggested that thalamic nuclei receive projections from the spinal cord (spinothalamic tract), cerebellum, area octavolateralis, and parietal organs (Heier, 1948; Nieuwenhuys, 1977). Thus, it appears that sensory information from many different sources is processed in the nucleus dorsalis thalami and then relayed to coordinating telencephalic centers, such as the striatum. Cells of the nucleus ventralis thalami are also retrogradely labeled after injections of FDA into the striatum, suggesting that they also project to this nucleus. Projections from the nucleus ventralis thalami appear to arise bilaterally, with the fibers crossing in the commissura postopticae. A small number of retrogradely labeled cells has recently been reported in the ventral thalamus of both anuran and urodele amphibians (see Marı́n et al., 1997a). Striatal dopaminergic afferents Previous results show that the striatum in lamprey receives a strong catecholaminergic innervation (Brodin et al., 1990b; Pierre et al., 1994). Here, we report that this innervation is mainly dopaminergic. In most vertebrates, this dopaminergic innervation originates mainly from the ventral mesencephalon. Because no catecholaminergic cells were found in the lamprey mesencephalon, those located rostrally to the dimesencephalic boundary in the posterior tubercle were supposed to correspond to the ventral tegmental area or substantia nigra of other vertebrates (Baumgarten, 1972). In the present study, DA-ir cells were found in the nucleus tuberculi posterioris, whereas no DA-ir cells were detected in the nucleus paratubercularis posterior, both of which contain TH-ir cells (Brodin et al., 1990b; Pierre et al., 1994; present results). The combination of retrograde tracing techniques (BDA) with TH immunohistochemistry demonstrated that some cells in the nucleus tuberculi posterioris that project to the striatum are dopaminergic. Catecholaminergic cells in the region of the posterior tubercle have also been identified in amphibians (Bartels, 1971; Wilczynsky and Northcutt, 1983; Wicht and Himstedt, 1988; González and Smeets, 1991; González et al., 1993, 1994a–c). This region, together with the medial part of the mesencephalic tegmentum, provides the dopaminergic innervation of the amphibian striatum (González et al., 1994c; Marı́n et al., 1997b). The catecholaminergic cells of the posterior tubercle in lamprey were regarded by Baumgarten (1972) as part of a structure that migrates into the base of the midbrain in tetrapods. Such a migration of cells has not yet been clearly demonstrated. The rostrocaudal gradient in the time of appearance of the posterior tubercle/midbrain TH-ir/DA-ir cells in amphibians during development favors this hypothesis (González et al., 1994a,b, 1995). This developmental pattern of a single merged cell group is also consistent with the embryonic origin of the substantia nigra (A9) and the ventral tegmental (A10) nuclei in mammals (see Fallon and Loughlin, 1985). The caudal portion of the striatum is less densely innervated by dopaminergic axons. Baumgarten (1972), however, described a few small, fluorescent, bipolar CSFcontacting cells with weakly fluorescent axons arborizing locally within the subependymal fiber layer. By using the streptavidin-biotin procedure, we also found a few weakly 84 M.A. POMBAL ET AL. Figure 7 STRIATAL AFFERENTS IN LAMPREY stained DA-ir cells in this region that appeared to make contact with the surface of the ventricle. Striatal afferents from the hypothalamus The injections of FDA into the striatum revealed large numbers of retrogradely labeled cells in both the ipsi- and contralateral hypothalamus. Considerable numbers of cells of both CSF and non-CSF-contacting types were labeled in the ipsilateral hypothalamus, whereas only occasional cells were labeled on the contralateral side. Part of the dorsal hypothalamus where 5-HT (Brodin et al., 1990b; Pierre et al., 1992), TH (Pierre et al., 1994; present results), and DA (present results) immunoreactivities are found may correspond to the paraventricular organ (PVO) of gnathostomes (Pierre et al., 1994; Pombal and Puelles, unpublished results). It has been suggested that the PVO of the hypothalamus of amphibians and reptiles may provide a massive monoaminergic innervation of the striatum (Sims, 1977; Tohyama et al., 1977; Dubé and Parent, 1982; Wilczynski and Northcutt, 1983; Dubé et al., 1990). However, recent studies with double-labeling combining tract-tracing and immunohistochemistry for DA or TH have demonstrated that the PVO does not project to the striatum in either anurans or urodeles (González et al., 1994c; Marı́n et al., 1997b). The same experiments also revealed striatal afferent cells in the ventral hypothalamus in these two species (González et al., 1994c; Marı́n et al., 1997a). Our results show that nondopaminergic hypothalamic structures also project to the striatum in lampreys. Striatal afferents from the olfactory bulb, preoptic region, and pallium Some retrogradely labeled cells were present medially in the olfactory bulb after injection of FDA into the striatum. The presence of olfactory bulb afferents to the striatum is in agreement with previous reports of labeled axonal terminals in the striatum after HRP or HRP-WGA injections into the olfactory bulb (Northcutt and Puzdrowski, 1988; Polenova and Vesselkin, 1993). Striatal projections from the olfactory bulb (Northcutt and Royce, 1975; Dubé Fig. 7. Photomicrographs of examples of DA-ir cells and fibers in the mediobasal diencephalon of the river lamprey. A,B: Transverse sections showing numerous DA-ir CSF-contacting cell bodies in the nucleus dorsalis hypothalami. Most labeled fibers course lateroventrally and then turn rostrally or caudally. C: Sagittal section illustrating the dopaminergic innervation in diencephalon (D), mesencephalon (M), and rostral rhombencephalon (R). DA-ir fibers, mainly originating from the ventral diencephalon, course caudally to innervate the brainstem. Note also the strong dopaminergic innervation of the neurohypophysis (arrow). D: Sagittal section through the ventrocaudal hypothalamus showing numerous CSF-contacting DA-ir cells in the nucleus commissurae postinfundibularis and nucleus ventralis hypothalami (arrow). Note also that not all cell bodies surrounding the recessus posterior (star) are immunopositive. A dense dopaminergic plexus appears caudally to this recessus. E: Transverse section showing numerous CSF-contacting cells in the nucleus commissurae postinfundibularis, surrounding the recessus posterior (rpos). F: Horizontal section showing CSF-contacting DA-ir cells in the nucleus ventralis hypothalami (vhy) and non-CSF-contacting cells in the nucleus tuberculi posterioris (ntp). Note also the presence of a dense dopaminergic lateral plexus, whereas the decussation of the fasciculus retroflexus (f) is free of labeled fibers. For other abbreviations, see list. Scale bars 5 400 µm in A,B, 500 µm in C, 200 µm in D–F. 85 et al., 1990; González et al., 1994c; Marı́n et al., 1997a) have also been reported in amphibians. The preoptic region provides afferents to the striatum. The preoptic area has also been shown to be reciprocally connected with the striatum via the medial forebrain bundle in amphibians (Wilczynski and Northcutt, 1983; Dubé et al., 1990; Marı́n et al., 1997a). Although this region, together with the suprachiasmatic nucleus, was suggested as another possible source of the dopaminergic innervation of the amphibian striatum, recent doublelabeling experiments did not support this hypothesis (González et al., 1994c; Marı́n et al., 1997b). Large numbers of cell bodies were retrogradely labeled in both the dorsal and the ventral parts of the ipsilateral lateral pallium after injections into the striatum, and a few were found in the ipsilateral, medial, and dorsal pallium, suggesting that all major pallial divisions project to this nucleus. Injections of HRP, HRP-WGA, or DiI in the lateral and medial pallium also demonstrated efferents to the striatum (Polenova and Vesselkin, 1993; Northcutt and Wicht, 1997). Although the latter authors described terminals from the medial pallium bilaterally in the striatum, only ipsilateral efferents were observed in the present study. Retrogradely labeled cells in the medial pallium of amphibians (González et al., 1994c; Marı́n et al., 1997a) and the lateral pallium of Caiman crocodilus (Brauth and Kitt, 1980) have also been described after injections of different tracers into the striatum. Striatal afferents from the rhombencephalon In the present study, some retrogradely labeled cells were found in the ipsilateral isthmus region, in the presumed locus coeruleus, and also in the raphe region, just ventral to the trigeminal motor nucleus and close to the midline. In amphibians, a few cells located in the midbrain tegmentum, locus coeruleus, parabrachial nucleus, raphe nuclei, different reticular nuclei, and the nucleus tractus solitarii give rise to ascending fibers that reach the striatum region (González et al., 1994c; Smeets et al., 1995; Marı́n et al., 1996, 1997a,b). In reptiles, cells of the nucleus tegmenti pedunculopontinus, locus coeruleus, and the raphe system also project to the ventrolateral area of the telencephalon, which is a presumed homologue of the mammalian striatum (Brauth and Kitt, 1980). In birds, some neurons located near the midline of the caudal midbrain tegmentum (Wild, 1987) and within the locus coeruleus, nucleus subcoeruleus dorsalis, and nucleus subcoeruleus ventralis (Kitt and Brauth, 1986) project to the paleostriatum. In mammals, the dorsal raphe nucleus (Steinbusch et al., 1981a) and probably also the locus coeruleus (Parent et al., 1983) provide inputs to the striatum. Some double-labeled cells were found in the isthmic region after BDA application in the striatum in combination with TH immunohistochemistry, which, thus, can be considered homologous to the locus coeruleus of amniotes. Similar results have also recently been reported in amphibians (Marı́n et al., 1996, 1997b). No structures caudal to the raphe region were found to project to the lamprey striatum. Even in our best experiment, only a few cells were retrogradely labeled in the raphe region after 65 hours of tracer transport in vitro (see above). Thus, we cannot rule out the existence in lamprey of other caudally located structures projecting to the striatum, like the nucleus tractus solitarii, where some Figure 8 STRIATAL AFFERENTS IN LAMPREY 87 TABLE 1. Comparative Analysis of the Catecholaminergic System in the Lamprey Brain and Spinal Cord and of the Projections to the Striatum1 T D M R SC Cell populations igl noa nca npo ncpo dth hy ncpi ntp2 ncp ot arn3 vn nts cc Vcc Lcc TH-ir (1) TH-ir (2) DA-ir (2) DA-ir (3) st (4) 1 1 1 2 1 1 2 2 2 2 2 2 1 2 1 1 1 1 2 1 1 1 1 2 1 1 2 2 2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 6 2 2 2 2 1 2 2 1 1 1 2 1 2 2 1 2 2 1 2 1 2 0 1 1 1 2 2 2 2 1 2 2 2 2 1 2 2 1The presence (1) or absence (2) of immunoreactivity (-ir) or inputs is indicated in the different regions obtained from the results of 1) Pierre et al. (1994; adult river lamprey), 2) present study, 3) Yáñez et al. (1992; larval sea lamprey), and 4) present study. different cell populations were described in ntp, both were immunoreactive to tyraxine hydroxylase (TH) but only one to dopamine (DA; see Discussion). 3Includes both nucleus isthmi rhombencephali dorsalis posteior and nucleus isthmi rhombencephali ventralis medialis (which includes the presumed locus coeruleus) of Pierre et al. (1994). 6, Only weakly dopamine (DA) ir cells were detected by using streptavidin-biotin (SAB) complex; 0, no cells were retrogradely labeled in this nucleus following injection of fluorescein-coupled dextran-amine (FDA) or BDA in striatum. Lcc, lateral to the central canal; Vcc, ventral to the central canal. The different regions are defined in the list of abbreviations. 2Two DA-ir cells were also located (present results), or reticular nuclei. Dopaminergic system in cyclostomes The general distribution pattern of DA-ir neurons in the adult river lamprey brain, in general, is similar to that of TH-ir cells reported in the same species (Pierre et al., 1994), but several differences deserve a comment (see Table 1). 1) Some cell populations (the nucleus olfactorius anterior, nucleus dorsalis thalami, pretectum, and nucleus isthmirhombencephali dorsalis posterior) were positive for TH (Pierre et al., 1994) but not for DA or TH (present study). This difference might be related to the use of different TH antibodies. Another possibility is that L-3,4Dihydroxyphenylalanine (L-DOPA) could be the biologi- Fig. 8. Dopamine-immunoreactive cells and fibers in the brainstem and rostralmost spinal cord of the river lamprey. A: Transverse section through the lamprey tectum opticum showing some small dopaminergic cell bodies (arrows) in the superficial stratum (ss). Note also the presence of DA-ir fibers in the periventricular (ps) and the central strata (cs). B: Transverse section through the isthmic region showing some weakly DA-ir cell bodies (arrows) in the ventromedial part of the isthmic nuclei as well as numerous immunopositive fibers. C: Transverse section at the level of the nucleus motorius nervi trigemini showing the dopaminergic innervation at this level. Numerous immunopositive fibers are distributed in the ventromedial rhombencephalic tegmentum, whereas the region of trigeminal motoneurons (Vm) is devoid of labeled fibers. In the octavolateral area, both dorsal (dn) and medial (mn) nuclei are clearly delineated by dopaminergic fibers, whereas some fibers are located in the ventral nucleus of this area, where two small dopaminergic cell bodies (small arrows) are also present. A few labeled fibers are also intermingled within the radix sensitivus nervi trigemini (dV). D: Magnification of a parallel section showing a dopaminergic cell (arrow) in the ventral nucleus of the octavolateral area (vn), just over the rostralmost part of the radix sensitivus nervi trigemini (dV). Note also the presence of dopaminergic fibers in the radix sensitivus nervi trigemini (dV). E: Transverse section through the caudal rhombencephalon showing small CSFcontacting DA-ir cells (small straight arrows) in the midline (sulcus medianus) and slightly displaced as well as one DA-ir cell in the presumed nucleus tractus solitarii (large straight arrow). Note also the rich dopaminergic ventromedial plexus originating from the small CSF-contacting cells as well as the pattern of innervation in the rhombencephalic tegmentum. The curved arrow indicates one root of the nervi vagi. F: Transverse section of the rostralmost spinal cord showing DA-ir cells contacting the central canal as well as some non-CSF-contacting cells (small solid arrows) displaced ventrally or laterally. Numerous dopaminergic fibers descending from the brain distributed in the dorsal and dorsolateral parts of the spinal cord. A dense dopaminergic plexus originating from intrinsic dopaminergic cells is evident in the ventromedial part of the section. The large open arrow points to a spinal ventral root. For other abbreviations, see list. Scale bars 5 100 µm for A,B,D,E, 300 µm in C, 400 µm in F. cally active catecholamine, as suggested in some cell populations in the mammalian hypothalamus (Okamura et al., 1988; Vincent and Hope, 1990; Mons et al., 1991), in reptiles (Smeets and Steinbusch, 1990) and in amphibians (González and Smeets, 1994). 2) Other cell populations (the nucleus commissurae anterior, superficial stratum of the tectum opticum, ventral nucleus of the octavolateral area, and some non-CSFcontacting cells located ventral and lateral to the spinal central canal) were positive for DA but not for TH. It is possible that the low level of TH in these small cell populations cannot be detected with these techniques or that these are DA-accumulating cells that do not synthesize this amine, as pointed out by Smeets and González (1990). Similar discrepancies between the distribution of DA-containing cells and TH-ir cells have been observed in different cell populations of other vertebrates (for review, see Manso et al., 1993). 3) In the region of the posterior tubercule, the distribution and size of DA-ir and TH-ir (Brodin et al., 1990b; Pierre et al., 1994; present results) appears to be different. The small cells located in the nucleus tuberculi posterioris were both DA-ir and TH-ir, whereas the larger cells in the nucleus paratubercularis posterior were only TH-ir. There are two recent studies on the catecholaminergic innervation of the hagfish brain. Presumed catecholaminergic cells were found in the preoptic region, the hypothalamus (CSF- and non-CSF-contacting types), and the isthmal region of Eptatretus stouti by using a TH antibody (Wicht and Northcutt, 1994). DA-ir cells of both CSF- and non-CSF-contacting types were found immunohistochemically only in the hypothalamus of Eptatretus burgeri (Kadota et al., 1993). Curiously enough, these results are in agreement with the distribution of DA-ir cells in the larval lamprey, where most immunopositive cell bodies are located in the hypothalamus and have apical dendrites that reach the surface of the ventricle (Table 1; Yáñez et al., 1992). Although DA1 fibers are widely distributed throughout the brain and spinal cord, it appears that the dopaminergic system in hagfishes is much more restricted than in adult lampreys (present results). Comparison with other vertebrates A recent review of the phylogenetic and developmental aspects of the catecholaminergic systems in the vertebrate central nervous system (Smeets and Reiner, 1994b) revealed that most catecholaminergic cell groups are present in all species studied so far. The present results demon- 88 M.A. POMBAL ET AL. Fig. 9. Photomicrographs of double-labeled cells following BDA injection into the left striatum combined with indirect immunofluorescence for tyrosine hydroxylase (TH). A,B: Horizontal section through the nucleus tuberculi posterioris showing two double-labeled cells (arrows in A, TH-ir cells; arrows in B, BDA retrogradely labeled cells). C,D: Transverse section through the isthmic region showing two double-labeled cells in the presumed nucleus coeruleus (arrows in C, TH-ir cells; arrows in D, BDA retrogradely labeled cells). Scale bar 5 100 µm. strate that the lamprey brain also has a well-developed dopaminergic system. In addition to this general scheme, some groups of catecholaminergic cells were found in the present study but were not observed previously: 1) A limited number of small DA-ir cells are present in the superficial stratum of the tectum opticum (present results). Small TH-ir cells were also identified in the most superficial and rostral intermediate layers of the tectum opticum in Heterodontus and Squalus, respectively (Stuesse et al., 1994). 2) Some small DA-ir cells are also present in the boundary between the ventral nucleus of the octavolateral area and the descending trigeminal tract. Catecholaminergic cells have been described in a similar position in Apteronotus (see Meek, 1994), which was referred to as nucleus of the descending trigeminal tract. 3) A CSF-contacting DA-ir cell group is present in the midline of the caudal lamprey brainstem. DA-ir ependymal and subependymal cells were also found in the same location in Rana and Pleurodeles by González and Smeets (1991). This dopaminergic cell population was thought to represent a derived character of amphibians, because it had not been observed in any other vertebrate. 3) In the lamprey spinal cord, in addition to CSFcontacting cells described in the spinal cord of most vertebrates (Pierre et al., 1994; Smeets and Reiner, 1994b; Schotland et al., 1995; present results), some non-CSFcontacting, DA-ir cells are also present ventrally and slightly lateral to the central canal (Schotland et al., 1995; present results). It has also been demonstrated that these cells colocalize 5-hydroxytryptamine (5-HT; Schotland et al., 1995; Zhang et al., 1996), which has additive effects in their action as modulators of spinal neurons controlling locomotion (Schotland et al., 1995). STRIATAL AFFERENTS IN LAMPREY Other features of the dopaminergic system in the river lamprey The particular location of the CSF-contacting DA-ir cells in the preoptic region, overlying the chiasma opticum, clearly resembles that of the dopaminergic suprachiasmatic cell population of other vertebrates (for review, see Smeets and Reiner, 1994b). This dopaminergic cell group, labeled here as the nucleus commissurae anterior, also contains CSF-contacting cells in the recessus praeopticus. Thus, it could be the homologue of the anterior preoptic region of other vertebrates. An important feature is the high number of DA-ir CSF-contacting cell bodies in the hypothalamus of lampreys compared with other vertebrates. This finding, together with observations made in amphibians (González and Smeets, 1994) and reptiles (Smeets, 1994), supports the notion that there is a reduction of CSF-contacting catecholaminergic cells from fishes to mammals (Parent et al., 1984). The mesencephalic dopaminergic cell group, including the retrorubral (A8), substantia nigra (A9), and ventral tegmental area (A10), has not been observed in cyclostomes (Kadota et al., 1993; Pierre et al., 1994; present results), holocephalians, and actinopterygian fish (Smeets and Reiner, 1994b). A correlation throughout phylogeny between a decreased PVO in the diencephalon and an increase in the number and distribution of monoamine neurons within the mesencephalon was suggested (Parent et al., 1984). Although the available data in lampreys support this correlation, recent data showing numerous TH-ir cell bodies in the hypothalamus of both anamniotes and amniotes, together with well-developed dopaminergic cell groups in the midbrain (Smeets and Reiner, 1994b), do not appear to support such a notion. CONCLUSIONS The striatum in lamprey receives inputs from different regions of the brain and a dense dopaminergic innervation that originates from the nucleus tuberculi posterioris in the diencephalon. The overall dopaminergic innervation of the lamprey brain shows several striking similarities with the general vertebrate scheme; however, it also presents some differences. The largest number of DA-ir cells occurs in the hypothalamus, which is a common feature of the dopamine system in fishes and amphibians, whereas no DA-ir cells were found in the midbrain tegmentum, which represents the most prominent catecholaminergic population of amniotes. ACKNOWLEDGMENTS We thank Drs. H.W.M. Steinbusch and T. Hökfelt for generous supply of DA and TH antisera and Drs. L. Brodin, D. Parker, and P. Wallén for helpful comments on the paper. We also thank H. Axegren, M. Bredmyr, and M. Podal for skilful technical assistance. 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