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Please be advised that this information was generated on 2017-12-05 and may be subject to change. THE JOURNAL OF COMPARATIVE NEUROLOGY 363:197-220 (1995)
Anuran Dorsal Column Nucleus: Organization, Immunohistochemical Characterization, and Fiber Connections in Rana perezi and Xenopus
A. MUÑOZ, M. MUÑOZ, A. GONZALEZ, an d HJ. TEN DONKELAAR Department of Cell Biology, Universidad Complutense de Madrid, 28040 Madrid, Spain (A.M., M.M., A.G.), and Department of Anatomy and Embryology, University of Nijmegen, 6500 HB Nijmegen, The Netherlands (A.M., H.J.T.D.)
ABSTRACT As part of a research program on the evolution of somatosensory systems in vertebrates, the dorsal column nucleus (DCN) was studied with (immimo)histochemical and tract-tracing techniques in anurans (the large green frog, Rana perezi, and the clawed toad, Xenopus laevis). The anuran DCN contains some nicotinamide adenine dinucleotide phosphate diaphorase- positive neurons, very little calbindin D-28k, and a distinct parvalbumin-positive cell popula tion. The anuran DCN is innervated by primary and non-primary spinal afferents, by primary afferents from cranial nerves V, VII, IX, and X, by serotonin-immunoreactive fibers, and by peptidergic fibers. Non-primary DCN afferents from the spinal cord appear to arise throughout the spinal cord, but particulai’ly from the ipsilateral dorsal gray. The present study focused on the efferent connections of the DCN, in particular the targets of the medial lemniscus. The medial lemniscus could be traced throughout the brainstem and into the diencephalon. Along its course, the medial lemniscus gives off collaterals to various parts of the reticular formation, to the octavolateral area, and to the granular layer of the cerebellum. At mesencephalic levels, the medial lemniscus innervates the lateral part of the torus semicircularis as well as various tegmental nuclei. A striking difference between the two species studied is that while in R. perezi medial lemniscal fibers do not reach the tectum mesencephali, in X. laevis, intermediate and deep tectal layers are innervated. Beyond the midbrain, both dorsal and ventral thalamic areas are innervated by the medial lemniscus. The present study shows that the anuran 4‘lemniscal pathway” is basically similar to that of amniotes. © 1995 Wiley-Liss, Inc.
Indexing terms: amphibians, somatosensory system, medial lemniscus, thalamus, torus semicircularis
In terrestrial vertebrates, two basic systems of ascending by dorsal root fibers from lumbar and thoracic segments, spinal projections are found (Willis and Coggeshall, 1991): whereas those of the cervical enlargement project to the 1) a primary afferent ascending spinal projection via the lateral (“cuneate”) compartment (Antal et al., 1980; dorsal funiculus to the dorsal column nucleus, giving rise to Nikundiwe et al., 1982; Jhaveri and Frank, 1983). In the medial lemniscal pathway to the thalamus; and 2) a Xenopus laevis a non-primary afferent projection to the secondary afferent projection via the lateral funiculus (i.e., DCN or postsynaptic dorsal column system was also demon the spinal lemniscus) to the reticular formation, mesen strated (ten Donkelaar and de Boer-van Huizen, 1991). In cephalon, and thalamus. ranid frogs, Vesselkin and co-workers (Vesselkin et al., In anurans, anterograde degeneration studies (e.g., Ebbes- 1971; Vesselkin and Kovacevic, 1973), Silvey et al. (1974), son, 1969, 1976; Hayle, 1973a,b) did not demonstrate a and Neary and Wilczynski (1977) described a contralateral spinothalamic tract, and the existence of a dorsal column- projection of the DCN (or perisolitary band) to thalamic medial lemniscal system remained a much debated question nuclei. More recent cobalt labeling studies in Rana escu- until the early 1980s. A recent anterograde tracer study showed a distinct direct spinothalamic projection in an Accepted May 25,1995. urans (A. Muñoz et al., 1994). The anuran dorsal column Address reprint requests to Dr. H.J. ten Donkelaar, Department of nucleus (DCN) is so mato topic ally arranged in such a fash Anatomy and Embryology, Faculty of Medical Sciences, University of ion that its medial (‘‘gracile”) compartment is innervated Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
© 1995 WILEY-LISS, INC 198 A* MUÑOZ ET AL. lenta (Antal et al., 1980; Urbán and Székely, 1982) and are available for anurans. Therefore, the existence of horseradish peroxidase (HEP) tracing in Rana perezi (M. different cell populations within the anuran dorsal column Muñoz et aL, 1991) and X. laevis (ten Donkelaar and de nucleus was studied using nicotinamide adenine dinucleo Boer-van Huizen, 1991; A. Muñoz et al., 1993) suggest that tide phosphate (NADPH) diaphorase (d) histochemistry the amphibian DCN-medial lemniscal system in fact might (NADPHd being a marker for NOS), calbindin D-28k, closely resemble the system found in amniotes, With electro- parvalbumin, GAB A, and glycine immunohisto chemistry. physiological techniques, Urbán and Székely (1982) showed Additionally, data on the serotonergic and peptidergic a rather extensive, contralateral thalamic somatosensory innervation of the DCN will be discussed. It will be shown projection following stimulation of either the second dorsal that the anuran dorsal column-medial lemniscus system is root, the dorsal funiculus, or the dorsal column nucleus. basically similar to that of amniotes. The anuran medial lemniscus also innervates the lateral part of the torus semicircularis (Comer and Grob stein, 1981; Wilczynski, 1981; Neary and Wilczynski, 1986). MATERIALS AND METHODS The present study is part of a research program on the The animals (60 adult specimens of R . perezi and 45 evolution of somatosensory systems in vertebrates. The young adult X laevis) were obtained from laboratory stock development, chemical neuroanatomy, and circuitry of of the Department of Cell Biology, University Complutense somatosensory systems is being studied in amphibians, of Madrid (R. perezi) and the Department of Animal urodeles as well as anurans. In the present study, the Physiology, University of Nijmegen (X. laevis). For a cytoar- connectivity of the dorsal column nucleus in two anuran chitectonic analysis of the obex region, Nissl (cresylecht amphibians, the Spanish green frog, R . perezi (formerly R. violet)-stained series of both anurans were available, cut ridibunda) and the clawed toad, X. laevis, was analyzed, either trans ver sally, horizontally, or sagittally at a thick using mainly HRP and bio tiny 1 at ed dextran amine (BDA) ness of 20 jxm. Adjacent sections were stained with silver tracing techniques. Little is known about the chemical proteinate, according to either Bodian’s (1936) or Klüver neuroanatomy of the anuran DCN. The mammalian cune- and Barrera’s (1953) technique. The histochemical, immu- ate and gracile nuclei are characterized by the presence of nohistochemical, and tract-tracing techniques used in this 7-aminobutyric acid (GABA)ergic (inter)neurons, and are study are discussed below. The nomenclature used is based innervated by substance P-positive and many other peptider on studies by Opdam et al. (1976) and Nikundiwe and gic fibers (see Rustioni and Weinberg, 1989, for review). Nieuwenhuys (1983) on the brainstem, and by Neary and Recent studies (e.g., Celio, 1990; Rausell and Jones, 1991a,b; Northcutt (1983) on the anuran diencephalon. Rausell et al., 1992; Menétrey et al., 1992a,b; Maslany et al., 1992; Ren and Ruda, 1994) also showed a certain preferen tial distribution of calcium-binding proteins like calbindin NADPH-diaphorase histochemistry D-28k and parvalbumin for somatosensory structures in Four adult frogs (R. perezi) were anesthetized in a 0.3% cluding the dorsal column nuclei. Nitric oxide synthase solution of tricaine methanesulphonate (MS222, Sandoz) (NOS) possibly marks a population of local circuit neurons and subsequently perfused trans car dially with a 0.9% within the DCN (Valtschanoff et al., 1993). No such data saline solution followed by a fixative containing 4% parafor
Abbreviations
A anterior thalamic nucleus nV trigeminal nerve Ad anterodorsal tegmental nucleus nVII facial nerve Av anteroventral tegmental nucleus nVIII vestibulocochlear nerve bv blood vessel nIX glossopharyngeal nerve C central thalamic nucleus nX vagal nerve c caudal P posterior thalamic nucleus cb cerebellum PDCS postsynaptic dorsal column system cdrg cervical dorsal root ganglion P r principal nucleus of the torus semicircularis cho chiasma opticum preg preotic ganglion DCN dorsal column nucleus Ptg pretectal gray df dorsal funiculus ptrg pretoral gray DH dorsal hypothalamic nucleus r rostral dh dorsal horn Rai nucleus raphes inferior dlf dorsolateral funiculus rf reticular formation dr3 third dorsal root Hi nucleus reticularis inferior dth dorsal thalamus Rm nucleus reticularis medius Ep posterior entopeduncular nucleus Rs nucleus reticularis superior Hd dorsal habenular nucleus SC suprachiasmatic nucleus Hv ventral habenular nucleus sol solitary tract Is nucleus isthmi tm tectum mesencephali IXm motor nucleus of the glossopharyngeal nerve tor torus semicircularis iz intermediate zone TP nucleus of the posterior tubercle La lateral thalamic nucleus: anterior division trVds descending tract of the trigeminal nerve Lam laminar nucleus of the torus semicircularis VH ventral hypothalamic nucleus ldrg lumbar dorsal root ganglion vh ventral horn 1m lemniscus medialis yds nucleus of the descending tract of the trigeminal nerve Lpd lateral thalamic nucleus: posterodorsal division Vllld descending nucleus of the nerve VIII Lpv lateral thalamic nucleus: posteroventral division VHIv ventral nucleus of the nerve VIII Mag magnocellular nucleus of the torus semicircularis Vld ventrolateral thalamic nucleus: dorsal part Mg magnocellular preoptic nucleus Vlv ventrolateral thalamic nucleus: ventral part Nsol nucleus of the solitary tract VM ventromedial thalamic nucleus NPv nucleus of the periventricular organ Vm motor trigeminal nucleus THE ANURAN DORSAL COLUMN NUCLEUS 199 maldehyde and 15% saturated picric acid in 0.1 M phos GABA immunohistochemistry (eight cases). After col phate buffer (pH 7.4). The brain and spinal cord were taken chicine (Sigma) injections (3 fjil, containing 20 |JLg/(Jil) into out and further fixed in the same fixative for 6-8 hours at the fourth ventricle of deeply anesthetized animals, and room temperature. They were subsequently immersed in a perfusion after survival times of 5-12 hours, the following 30% sucrose phosphate buffer solution at 4°C, embedded in procedure was used: 1) rabbit anti-GABA (Sigma), diluted a 15% gelatin and 30% sucrose solution, and stored for 5 1:1,000, overnight; 2) swine anti-rabbit (Nordic), diluted hours in a 4% formaldehyde solution at room temperature. 1:50, for 2 hours; and 3) PAP-rabbit (Dakopatts), diluted On a freezing microtome, 30 or 40 jxm frontal sections were 1:600, for 2 hours. cut and collected in phosphate buffer. Free-floating sections Glycine immunohistochemistry (ñve cases), 1) Rabbit were incubated in a medium containing 1 mM (3-NADPH, anti- glycine (Chemicon), diluted 1:200-1:500, for 24 hours; 0.8 mM nitroblue tetrazolium, and 0.06% Triton X-100 in 2) goat anti-rabbit (Sigma), diluted 1:50—1:200; and 3) 0.1 M phosphate buffer (pH 7.6) at 37°C for 1-2 hours. rabbit PAP-complex (Dakopatts), diluted 1:600, for 2 hours. After incubation, the sections were thoroughly rinsed in Calcitonin gene-related peptide (CGRP) immunohisto- phosphate buffer, mounted on gelatin-coated glass slides, chemistry (ñve cases). 1) Rabbit anti-CGRP (Amersham), and, after drying overnight, coverslipped. Selected sections diluted 1:2,000, for 12-24 hours; 2) swine anti-rabbit were counterstained with 1% cresyl violet. In two cases, the (Nordic), diluted 1:50, for 2 hours; and 3) rabbit PAP sections were also processed for tyrosine hydroxylase immu- complex (Dakopatts), diluted 1:600, for 2 hours. nohistochemistry after rinsing. Substance P (SP) (ñve cases) and Leu-enkephalin (L- Enk) immunohistochemistry (four cases). 1) Rabbit Immunohistochemical procedures anti-SP or rabbit anti-L-Enk (CRB), diluted 1:2,000, over For the immunohistochemical procedures used, animals night; 2) swine anti-rabbit (Nordic), diluted 1:50, for 2 were anesthetized with an overdose of MS222 and transcar- hours; and 3) rabbit PAP complex (Dakopatts), diluted dially perfused with saline followed by a mixture of 4% 1:600, for 2 hours. paraformaldehyde, 0.05% glutaraldehyde, and 0.2% picric Neuropeptide Y (NPY) immunohistochemistry (four acid in 0.1 M phosphate buffer (pH 7.4). The brain and cases). 1) Rabbit anti-NPY serum (gift from Dr, J.D. Mikkelsen), diluted 1:1,000, for 36 hours; 2) swine anti spinal cord were removed and postfixed for 4-7 hours in the rabbit (Nordic), diluted 1:50, for 1 hour; and 3) rabbit PAP same fixative, embedded in 15% gelatin with 30% sucrose complex (Dakopatts), diluted 1:800, for 1 hour. (R. perezi) or in polyacrylamide (.X. laevis). Brains were cut Serotonin (5~HT) immunohistochemistry (ñve cases). frontally on a freezing microtome or on a Vibratome at 40 1) Rabbit anti 5-HT (gift from Dr. H.W.M. Steinbusch), |im, and the sections w£re collected in a Tris-saline (TBS) diluted 1:1,000, overnight; 2) swine anti-rabbit (Nordic), buffer (0.05 M, pH 7.6). All antibodies were diluted in 0.1% diluted 1:50, for 2 hours; and 3) rabbit PAP complex normal serum of the species in which the secondary anti (Dakopatts), diluted 1:600, for 2 hours. body was raised, in TBS with 0.1% Triton X-100 (Sigma). In all cases, after rinsing, the sections were incubated The sections were preincubated for 1-2 hours in TBS with 0.5 mg/ml 3,3'-diaminobenzidine (DAB, Sigma) with containing 3% normal serum and 0.1% Triton X-100 and 0.01% H2O2 in phosphate buffer, for 10-15 minutes. After subsequently incubated in the primary antibody-containing another rinsing, the sections were mounted on glass slides solution for 24-36 hours at 4°C. Controls for the immuno- (mounting medium 0,25% gelatin in Tris buffer), dried histochemistry experiments included: 1) staining some overnight, and coverslipped. In most cases, visualization of selected sections with pre-immune mouse serum (1:1,000 the immunostaining was improved by processing the sec for tyrosine hydroxylase, calbindin D-28k, and parvalbumin tions with nickel-enhanced DAB (0.05% DAB, 0.01% H2O2, immunohistochemistry), or with rabbit serum (1:500 for 0.04% ammonium nickel sulphate in phosphate buffer). glycine; 1:1,000 for GABA, neuropeptide Y, and serotonin; p 1:2,000 for calcitonin gene-related peptide, substance P, Tract-tracing experiments and Leu-enkephalin immunohistochemistry) instead of the primary antibody (e.g., Fig. 5C); and 2) controls in which In vivo technique. All experiments were carried out primary antibody, secondary antibody, or the peroxidase- under surgical anesthesia with MS222. The following trac antiperoxidase complex were omitted. As an additional ers were used as retrograde and anterograde tracers: HRP control for the specificity of the labeling of calcium-binding (Boehringer), BDA, 10 kD (Molecular Probes D-1956), and proteins, some sections were stained using antibodies that rhodamine dextran amine (RDA; Molecular Probes D-1817). had been pre-adsorbed in an excess of parvalbumin or HRP was applied iontophoretically (a 15% HRP solution in calbindin. These procedures revealed a light, diffuse back phosphate buffer injected for 10 minutes using 5-8 pA ground. No stained cells or fibers were found in any of the positive pulsed current, 7 sec on/7 sec off), or as dry crystals cases. The sections were processed according to the peroxi- onto a fine tungsten needle, to the DCN (five cases), to the dase-antiperoxidase (PAP) technique (Sternberger, 1979) most lateral part of the torus semicircularis (two cases), or in a series of incubations with the following antisera: to the thalamus (three cases). BDA was recrystallized from Tyrosine hydroxylase (TH) immunohistochemistry (eight distilled water onto fine tungsten needles and applied to the cases), 1) Mouse anti-TH (Incstar), diluted 1:1,000, for proximal stumps of cut trigeminal nerves (four cases) or 24-72 hours; 2) goat anti-mouse (Nordic), diluted 1:100, for third spinal dorsal roots (three cases). Previous experi 3-5 hours; and 3) rat PAP complex (Incstar), diluted 1:500, ments (Nikundiwe et al., 1982, X. laevis; M. Muñoz et al. for 2 hours, 1991, R. perezi) in which HRP was applied to thoracic and Calbindin D~28k and parvalbumin immunohistochemis lumbar dorsal roots were used for the analysis of DCN try (12 cases). 1) Mouse anti-calbindin D-28k (Sigma) and projections of the more caudal dorsal roots. Additionally, mouse anti-parvalbumin (Sigma), diluted 1:1,000, for 24-72 material in which HRP or BDA was applied to the proximal hours; 2) goat anti-mouse (Nordic), diluted 1:100, for 3-5 stumps of the facial, glossopharyngeal, and vagal nerves hours; and 3) PAP, diluted 1:500, for 2 hours. could be analyzed. Alternatively, BDA was injected iontopho- 200 A. MUNOZ ET AL. retically as a 10% solution in phosphate buffer, into the 1980), HRP (Nikundiwe et al., 1982) or BDA (see Fig. 6) DCN (three cases), cervical dorsal horn (three cases), much more clearly delineates the DCN. cerebellum (three cases), and thalamus (four cases). Sur In X laevis, medial (“gracile”) and lateral (“cuneate”) vival times varied from 5 to 10 days. Subsequently, the compartments of the DCN can be distinguished above and animals were re-anesthetized and perfused through the lateral to the distinct solitary tract (Nikundiwe et al., 1982; heart with isotonic saline followed by a fixative containing Nikundiwe and Nieuwenhuys, 1983). A dorsal indentation 4% paraformaldehyde for the BDA experiments and 1.5% suggests such a subdivision. Medially, the DCN is difficult paraformaldehyde and 2% glu tar aldehyde for the HRP to distinguish from the nucleus of the solitary tract, and cases. The brain and spinal cord were removed, postfixed laterally it is very poorly segregated from the nucleus of the for 2-4 hours, and embedded in gelatin or polyacrylamide. descending tract of the trigeminal nerve. Both compart Sections were cut transversally or horizontally at 40 \xm on ments of the DCN consist of small (8-10 |xm) and medium a freezing microtome. Histochemistry for HRP followed the sized (15 fxm) multipolar elements. The medial, gracile part heavy metal intensification of the DAB-based HRP reaction begins at the level of the second spinal nerve and extends product (Adams, 1981). For visualizing BDA, an avidine biotin complex (Vectastain ABC Elite Kit, Vector Laborato into the brainstem, where it is situated dorsal and dorsolat ries) was used. Some BDA-reacted sections were rinsed and eral to the solitary tract (Fig. 1A,B). The lateral, cuneate further processed for calbindin D-28K or parvalbumin part extends further rostrally than the gracile part, and immunohistochemistry as described above. The black color borders on the nucleus of the descending trigeminal tract. of the BDA labeling contrasts with the calbinding protein In R. perezi, as in R. esculenta (Antal et al,, 1980), the labeling stained brown by using the DAB reaction without segregation of the DCN from the surrounding cell struc heavy metal intensification. RDA, re crystallized from dis tures such as the nucleus of the descending trigeminal tract tilled water onto sharp tungsten needles, was applied to the and the nucleus of the solitary tract is also rather poor. In thalamus and to the torus semicircularis. After survival R. perezi, the cell area dorsal and lateral to the solitary tract times of 2-4 days, animals were re-anesthetized with an only occasionally shows an indentation allowing the distinc overdose of MS222 and perfused with 0.1 M phosphate tion of medial and lateral compartments in the DCN (Fig, buffer (pH 7.4) followed by a fixative containing 4% parafor 1C-F). These gracile and cuneate subdivisions rostrally maldehyde in phosphate buffer. The brain and (rostral) extend to the level of the glossopharyngeal nucleus where spinal cord were taken out, embedded in polyacrylamide, they are slightly more laterally located since in that position left overnight in 15% saccharose in 0.1 M phosphate buffer, the most medial part of the rhomb encephalic alar plate is and cut transversally on a freezing microtome at 40 jxm. occupied by the caudal pole of the vestibular nuclear They were mounted in glycerin-gelatin and viewed with a complex and related descending vestibular root fibres (Fig. Zeiss fluorescence microscope with appropriate filter combi 1C,D). Lateral to the DCN cells, the cells in the dorsolateral nations. position of the alar gray are mingled with afferent fibers of In vitro technique. In ten young adult X laevist an in cranial nerves V, VII, IX, and X, forming the descending vitro approach was used, based on Cochran et al. (1987). tract of the trigeminal nerve. These dorsolateral alar gray The animals were deeply anesthetized with a 0.2% solution cells can be regarded as a component of the nucleus of the of MS222 and perfused with iced Ringer’s solution (75 mM descending trigeminal tract. NaCl, 25 mM NaHCOa, 2 mM CaCl2, 2 mM KC1, 0.5 mM MgCl2) 11 mM glucose; pH 7.4). The brains were removed, In X laevis and to a lesser extent also in R. perezi> the cell submerged in the same iced Ringer’s solution, and cut at area above and lateral to the solitary tract at the obex level middiencephalic or midmesencephalic levels. Applications is composed of two DCN compartments, the lateral of which of 3 kD BDA (Molecular Probes, D-7135), recrystallized at fades into the nucleus of the descending trigeminal tract. the tip of sharp tungsten needles, were made at the ventral Chemical neuroanatomy thalamus (five cases) or the torus semicircularis (five cases). NADPH-cliaphorase histochemistry. In the caudal part The brains were kept for 5-18 hours at room temperature of the rhomb encephalic alar plate in R. perezi, NADFHd- in continuously oxygenated Ringer’s solution (pH 7.4) with positive neurons were observed in the DCN, in the adjacent carbogen, and subsequently processed as described for the descending trigeminal nucleus, and in the nucleus of the in vivo BDA experiments. solitary tract (Fig. 2A,B). InX laevis, the caudal lateral line nucleus was labeled as well. In the DCN, a cluster of NADPHd-positive neurons was found, the dendrites of RESULTS which are directed dorsally toward the dorsal funiculus. It Delineation and (immuno)histochemical should be noted, however, that NADPHd-positive neuron characterization of the anuran populations are more distinct in the nucleus of the solitary dorsal column nucleus tract, mainly in its medial and ventromedial parts below the Cy to architecture. No distinct DCN or nucleus funiculi solitary tract, and in the nucleus of the descending trigemi dorsalis could be distinguished in most cytoarchitectonic nal tract (Fig. 2A,C). By combining NADPHd staining with studies of the anuran brain stem (e.g., Ariens Kappers and immunohistochemistry against TH, the NADPHd-positive Hammer, 1918; Zeehandelaar, 1921; Opdam et al., 1976). part of the nucleus of the solitary tract was shown to be Therefore, since Woodburne’s (1939) Marchi studies, the intermingled with the cat echo laminergic cells (Gonzalez anuran DCN is defined as the site of termination of dorsal and Smeets, 1991) situated ventral to the solitary tract. In funicular fibers in the caudal brainstem, rather than as a the caudal part of the rhomb encephalic alar plate of both cytoarchitectonic entity. Nevertheless, Nissl-stained sec anuran species studied, NADPHd-positive fibers were found tions of the brainstem at obex levels allow the delineation of predominantly in two bundles, i.e., the descending trigemi a DCN (Fig. 1), although labeling of spinal primary afferent nal tract and the solitary tract. In the solitary tract, projections to the obex level by cobalt staining (Antal et al., NADPHd-positive fibers were observed in its most dorsal %a
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' Ç 1 ■íf I m m : ï - llf m u - !ii: III f ( ; w* m ■ \ ' % '■:/J w-'J.l % & m . 4 m % v s v •V/.V • m á ; ; % * i p Ä ill ■■Sf ' V t %' mlM^&0Ï0§: 9 : iSív A. MUNOZ ET AL Fig. 8. Labeling observed after a BDA injection into the DCN of R. perezi (for injection site see also Fig. 10A). In a series of transverse sections through the diencephalon (A-D), brainstem (E—L), and spinal cord (M-O), the pattern of anterogradely labeled fibers and retrogradely labeled cells (black dots) is shown. Fig. 9. Labeling observed after a BDA injection into the DCN of X laevis. In a scries of transverse sections through the diencephalon (A-D), brainstem (E -M ), and spinal cord (N -P ), the pattern of anterogradely labeled fibers and retrogradely labeled cells (black dots) is shown. 210 A. MUNOZ ET AL eral to the isthmic nucleus. At caudal mesencephalic levels, lemniscus. In addition, a few cells were labeled bilaterally in the fibers turn dor sally along the lateral aspect of the the dorsolateral descending trigeminal tract. In in vitro midbrain, and most of them bend medially, where they BDA experiments in young adult X laevis a similar pattern terminate in the torus semicircularis (Figs. 8F,G, 9F,G, oflabelingwas observed (Fig. 12A,C). 10C,D). The principal, magnocellular, and commissural 2. Toral applications. When the injection sites were nuclei receive only a sparse DCN projection, but the limited to the torus semicircularis, neurons were retro laminar nucleus is densely innervated, mainly in its lateral gradely labeled within the DCN. They were particularly portion. A few fibers pass to the contralateral commissural found on the contralateral side, although an ipsilateral and principal nuclei of the torus semicircularis. Inii. perezi component was present as well. Two distinct cell groups medial lemniscal libers do not reach the mesencephalic were observed in R. perezi (Fig. 11B,E). The first one is tectum, while in X, laevis the intermediate and deep tectal made up of large cells with a minor component of small cells layers are innervated. These rather thick medial lemniscal located in the dorsalmost gray. They possess several pro fibers innervating the tectum mesencephali often give off cesses extending into the dorsal fiber layer. Their axons thin collaterals that terminate in the laminar nucleus of the course ventromedially, cross the midline, and form part of torus semicircularis (Figs. 9E-G, IOC). In both species, at the medial lemniscus. The second group of labeled cells is more rostral mesencephalic levels, the anterodorsal and located in the lateral marginal zone of the dorsal gray from anteroventral tegmental nuclei as well as the red nucleus the level of the obex to the second spinal segment. These are and the interstitial nucleus of the fasciculus longitudinalis large bipolar and irregular cells with long processes directed medialis are innervated by medial lemniscal fibers (Figs. mainly to the dorsal part of the lateral funiculus and into 8E,F, 9E,F). the dorsal funiculus, while their axons participate in the At rostral mesencephalic levels, scattered labeled fibers medial lemniscus. In addition, retrogradely labeled cells distribute to the pretoral gray, and, in X. laevis, also to the were always found in the ipsilateral descending nucleus of pretectal gray (Figs. 8E, 9E). Beyond the midbrain, both the trigeminal nerve following toral injections. After simi the dorsal and ventral thalamic areas are innervated by lar toral injections in X. laevis, retrogradely labeled cells in medial lemniscal fibers (Figs. 8A-D, 9A-D). A few thin, the DCN form a band positioned from dorsomedial to varicose fibers innervate the ventral parts of the posterior ventrolateral above the solitary tract. The most dorsally and central dorsal thalamic nuclei, whereas the ventrome located cells possess dendrites extending into the dorsal dial thalamic nucleus and the posterior tubercle are far funiculus, whereas more ventrolateral cells have dendrites more densely innervated. The fibers reaching the ventrome reaching the dorsal aspect of the lateral funiculus. Some dial nucleus pass through the dorsal and ventral parts of neurons were observed with dendrites reaching both the the ventrolateral thalamic nucleus and varicosities are also dorsal and dorsolateral funiculi (Fig. 12D). In in vitro found among its cells. Apart from a few fibers reaching the experiments, BDA was applied to the torus semicircularis of anterior nucleus of the dorsal thalamus (in two cases in R. X. laevis. The pattern of labeling in the DCN is shown in perezi), no labeling was found more rostrally in the anterior Figure 12B,D.E. diencephalon or in the telencephalon in any of the cases. 3. Cerebellar applications. In experiments with tracer 2. Extralemniscal ascending projections. Apart from the applications into the lateral portion of the cerebellar plate, medial lemniscus, the DCN gives rise to a distinct ipsilat- the underlying cerebellar nucleus and the adjacent gray eral ascending projection (Figs. 8I-K, 9I-L). Due to their were mostly implicated as well. At the obex region, these proximity, the ascending primary afferent spinal fibers applications resulted in the labeling of three cell groups. bypassing the injection site were most likely to be involved. Most labeled cells were found at the ventromedial margin of Such fibers are known to project to the octavolateral area the caudal extent of the fasciculus solitarius, on both sides and the cerebellum (Antal et al., 1980; Nikundiwe et al., of the medulla, probably due to the uptake of the tracer by 1982). Additionally, adjacent cell groups such as the nucleus fibers projecting from the nucleus of the solitary tract to the of the descending trigeminal nucleus and the nucleus of the nucleus visceralis secundarius (parabrachial region). In the solitary tract might have incorporated the tracer from the nucleus of the descending trigeminal tract labeled cells injection sites. Therefore, ipsilateral projections from the were found as well, mainly ipsilateral to the application site. DCN are difficult to demonstrate in anterograde experi The third group of retrogradely labeled cells was found, ments. bilaterally, in the DCN (Fig. 11C,F). These cells were Retrograde tracer experiments. To verify whether these mainly found ipsilaterally. Their axons seem to run to ascending projections really arise in the DCN, in both gether with the ascending primary afferent fibers from the anuran species injections of HRP, BDA, or RDA were placed spinal dorsal roots. into the thalamus, torus semicircularis, and cerebellar Efferent projections: descending. In experiments with region. BDA or HRP applications into the DCN region antero- gradely labeled axons could be followed from the injection 1. Thalamic applications. In this group of experiments, a site caudalwards into the spinal cord. These fibers course retrograde tracer was applied to the thalamus in such a way via the ipsilateral dorsal funiculus and form fine arboriza that both the dorsal and ventral thalamus were implicated. tions of thin varicose fibers terminating among the cells in Retrogradely labeled cells in the region of the DCN formed the dorsal horn throughout the cord, but particularly at a mixed population of irregular, large cells, and round, cervical levels. A sparse bilateral innervation of the interme small cells (Fig. 11A,D). Although the majority of the cells diate and ventral zones was also observed. These fibers were located contralaterally, a minor component of ipsilat could, however, represent fibers by-passing the injection eral cells was also present. The dendrites of these cells are site. Therefore, spinal injections with retrograde tracers long and directed both dorsally and ventro 1 aterally, reach were studied. A small population of cells, scattered in the ing the dorsal and the dorsolateral funiculi, respectively. area of the ipsilateral DCN, was always labeled after Their axons were followed into the contralateral medial injection of the various spinal segments (Fig. 10 G). ■if/roVi-J' r^ ,.v £ i j i w /ƒ % . ^ Ì %iff '% r * : # 9 ; * f 'Mi, m ¡KV{,- .....lilil 1 •'/(!■ !’ iiiiliif ili mM yyyfY.'S^'i vmM ■'■Y,’,' y/, - ■ ' ! / a : | ; i x l z 7\yM MrII ;y '.'.•'S'/'’ ■''yy,: ‘SY./’S’ ;V: mm 1§1§P^ I r>0ì& y /, 9®«» ■.’■Is , ■ /S//- i 'f m i ' w < / 0 '<\ ’/ :.\ '■'■'■ i l l Jllppgi,.,,,,,., .*v 1 1 M b l i l l f l p i ■:?x . M i: $§ i:7, i i i i 1* ■ y jh * "I i/if'. wM&m m t im l |I W; I s m m . m m s® ■ W, w \ b :!f : .4te: % •i* # t f > A y / - i i v.-! Mm ly ; w m f r y s . ’¿'■V 'W. :m. m ■y/, l i mi 'i'/; m p a '■■’■<>'<■: Mu s, M Milli « •'A- • I ■> ; m ••m- m M WMk M % - y . C * / < '* > m ■mi m •;oV: V/: >0, mii m i : : wm. > > V \ ÉWi « ■;s. « l i f c ■ ;/> , IMt i l i 10!). % * '■ M ■>/.' : t&MJM wm 'iMi.fi * \ i y ■ :. « 'Y y > . « < i x ? h iili: ^Ò^MÉ y / y SIM m s . ,v , 'S / i s , y . ' ’/ y " : ili ■■y\- :'r ■■’■:■ iitù r' 'i-y'yiM-. ■ y y ' y $ K , ••’ ,’• i W Z \ ■# iK:1 ■:''>■ ■ ' « ¡j 4'• -VViVj’A-. ’ ’ ’ , •fiVÀfy.v M ' i 'I ' I v| -"J i : T-1 : :’ y . :H:.Y-'y ’ • ' V ' - i y A J y y y y p . v v ^ y v ' Hp!^ « « i i1 ;V|, ƒy .:':'.’ ' .il'::';'.'.-'. ' ; < y ■ ;:’■>. v / i ai I'M : > v ^ > .v*ss. '■■ K< : ' w&m ; v - ; :.. ,-;: W0M- # : r ; r.-; l»toll’ ¡ ■ / / / ¿ k t y 'S S } ■;■:■,<■■■;■ ; : :.•:; : •: i * ; ^ ^ y.^ ^ r J r. ; ¡i 5 : f i * [ » '■ ’¿s ■ V i ; ' Mm ® M j; ij:!:! ¡ '< % V i- y ; - 5 S 5 g j i ■ ■ : V/VWa,,' •. ' A 1; IS1 . m m, 'M. p; ;Ì?i^ •M ISi M »V : • il ':< /•■ Ws is: IV; P ¡M ’> : ' '■ If W \.'/rw ri-O X Y;y''y,\- m IP m. I l i ! m W 8 & y * 4?- Hip m W': % Wf; liiiP y . \ ' . ' w . . ':7/ Ù . ì ' IMik lé iàii' •1 l i » :-.j.:V //; A'_; ; v . iv •:::^yyy.iy\\-.\ ;V< i i ■ ;S: : '; '< :: ' ì '-ì :a >:- ;i i ili Wì VMg-T m lillSIIIiifISI iH : - . W$h !l i ; ' ' y y y ■.■■'••■< :V :i; y % i :/ v '. : , \ ' . ' / / 1 < .'. -.1 Cf / - : ; ' c- : - % ' •: ; ' V < i MiV Ì l i ! y : > viX 'V K * '" ■ ...... , - / r ; , irnssism. '< :'•> •■.•/■'■ .'.'fi! < '):• ■ 'M . r- v .; ...... WdMM&'V V'. ■fi WÈ W ì vmm I 1 1 ■ .;///■ . ■m m. M l i m {&. w i i « ■‘m /Ì 8 : '¿i.V ' / / : ■ I'.'- t t - - . / / '■ V, »>//■Si' t . * # 8 t ■ j o ; - W:. # . : n i t ó f t : # . « i r ’.Crr.-../-:/.< t I f :■*< Mìèl m . '.•J V'. ■ •/'//' ■V •:V ' H i t , l i l i É S l l l i '///'.•'.< /. ' i l l • ' V 1/-' ‘ > -‘ Ù < i / . . 7 ' 'A*/: .Vf i ■ i'È b k w -Éfliilli«illÉIIIIiil - y \ ■yy.'i’K 'y/, 'MA: ...... '''/-, ìiÌÉlpfi wmmamm ' . 'i 'y j . III* r/< \ ' u - W é0ÈM ■ } y / ' \ r , ’; U l '<>.<■:■} I: m f i r ; 212 A. MUÑOZ ET AL. DISCUSSION that Calb-positive neurons form a major part of the solitary and trigeminal projection systems. In the trigeminal system In the present study the organization, immunohisto- of monkeys, both proteins are differentially expressed in the chemical characterization, and particularly the fiber connec ascending trigeminothalamic projections to the ventral tions of the anuran DCN were investigated. Although it is posteromedial (VPM) nucleus (Rausell and Jones, 1991a,b). obvious that the anuran DCN remains a rather ill-defined Antisera to parvalbumin and calbindin mark VPM rods and area in the caudal part of the rhombencephalic alar plate, matrix, which receive principal and spinal trigeminal input, and no selective markers for the DCN other than its respectively. A similar segregation has been demonstrated labeling by primary afferents from the spinal cord are for the ascending somatosensory projections from the available, NADPHd staining and immunohistochemical spinal cord (Rausell et al., 1992): A non-nociceptive Parv- staining of calcium-binding proteins and various neurotrans positive dorsal column-me dial lemniscal projection termi mitters certainly help in delineating and characterizing the nates in cytochrome oxidase (CO)-rich domains of the DCN. Since no clear cytoarchitectonic separation of the ventral posterolateral thalamic nucleus (VPL) where Parv- DCN into a medial, “gracile” nucleus and a lateral, “cune- positive neurons are found. Nociceptive Calb-positive spino ate” nucleus is obvious, the term dorsal column nucleus is thalamic fibers terminate in CO-poor domains of the VPL preferred. where Calb-positive cells are present (Rausell et al., 1992). The NADPHd histochemical technique, known to stain Against this background, the presence of Calb and Parv specific neurons (Thomas and Pearse, 1964), can selectively in the anuran DCN was studied. It appeared that in the alar stain particular populations of neurons in a Golgi-like plate of the caudal rhombencephalon, Calb-positive neu manner (Scherer-Singler et al., 1983). Throughout the rons were found particularly in the nucleus of the solitary brain, NADPHd and NOS localizations are identical (Bredt tract and in the descending nucleus of the trigeminal nerve, and Snyder, 1992). Therefore, NADPHd can be used as a continuing into the dorsal horn of the spinal cord. In both marker for NOS. Nitric oxide probably plays a major role as anuran species studied, hardly any Calb-positive neurons a neuronal messenger (Bredt and Snyder, 1992; Meller and were found in the DCN itself. This pattern of distribution of Gebhart, 1993; Schuman and Madison, 1994), The pres Calb-positive neurons suggests that in anuran amphibians, ence of NADPHd-positive cells and fibers in the mammalian as in mammals, Calb could be restricted to the nociceptive spinal cord (Valtschanoff et al., 1992) suggests that nitric part of the somatosensory system including neurons in the oxide may be involved in spinal sensoiy processing. In the dorsal horn of the spinal cord, and the descending nucleus rat DCN, Valtschanoff et al. (1993) found that most NOS- of the trigeminal nerve. In striking contrast, in both anuran positive neurons are also immunoreactive for GABA, but species a distinct Parv-positive DCN population was ob not for the excitatory transmitters glutamate and aspar served, particularly inX laevis. Parv-positive neurons were tate. Moreover, since NOS-positive neurons could not be found throughout the DCN, and their dendrites were labeled retrogradely from the thalamus or spinal cord, they mainly directed dorsally or laterally into the adjacent dorsal are probably local circuit neurons (Valtschanoff et al., funiculus. Immunostaining of Parv can therefore be used to 1993). In the anuran species studied, NADPHd-positive delineate the anuran DCN. It should be noted, however, neurons were found in- the DCN, but especially in the that the pattern of distribution of calcium-binding proteins adjacent nucleus of the solitary tract and the descending in the rat DCN is quite different. Maslany et al, (1992) nucleus of the trigeminal nerve, in keeping with data in found both Calb- and Parv-positive neurons in the cuneate mammals (e.g., Leight et al., 1990; Vincent and Kimura, and gracile nuclei, although Parv-positive DCN cells were 1992; Dohrn et al., 1994; Takemura et al., 1994). Since no more numerous. The distribution of Parv cells appeared to double-labeling studies for GABA or excitatory transmit be similar to the known distribution of thalamic projection ters were carried out, it remains to be analyzed whether neurons. these NADPHd-positive neurons are local circuit neurons In mammals, the presence of small GABAergic interneu or give rise to efferent projections such as the medial rons within the DCN has been extensively described (for lemniscus. reviews see Mugnaini and Oertel, 1985; Rustioni and Calcium-binding proteins such as Calb, calretinin, and Weinberg, 1989). Also glycinergic inhibitory effects within Parv are found in certain subpopulations of neurons in the the DCN were observed. Does the anuran DCN contain central and peripheral nervous system (Baimbridge et al., GABAergic inter neurons? In the present study small, round 1982; Garcia-Segura et al., 1984; Braun, 1990; Celio, 1990; or oval-shaped GABA-immunoreactive neurons were ob Ren and Ruda, 1994)* They even label entire pathways, and served. The pattern of labeling in other parts of the brain sometimes whole functional systems (Celio, 1990; An- stem is comparable to that described by Franzoni and dressen et al., 1993). In mammals, calcium-binding pro Morino (1989, R. esculenta), who, unfortunately, did not teins like calbindin and Parv show a preferential distribu include the most caudal part of the brainstem in their tion in somatosensory structures, including the DCN (e.g., analysis. Double-labeling studies, i.e.3 combinations with Celio, 1990; Rausell and Jones, 199la,b; Rausell et al, tract tracing or NADPHd, are needed to demonstrate 1992; Menetrey et al., 1992a,b; Maslany et al., 1992; Ren whether these GABA-immunoreactive neurons in the an and Ruda, 1994). Parv appears to be abundant in the uran DCN are actually interneurons. In this respect, it pathway for epicritic sensibility, i.e., the dorsal column- should be noted that Pritz and Stritzel (1989a) suggested medial lemniscal system, and Calb occurs in the whole taste that the reptilian (Caiman crocodilus) DCN lacks glutamic pathway of rats (Celio, 1990). In rats, Calb-positive neurons acid decarboxylase (GAD)-immunoreactive neurons, indicat are found in certain laminae of the dorsal horn (Antal et al., ing that the reptilian DCN—like the dorsal thalamus (see 1990; Ren and Ruda, 1994) including the cells of origin of Pritz and Stritzel, 1988)—lacks local circuit neurons. A few ascending spinal projections (Menétrey et al., 1992b), in the ;lycinergic neurons were found at the ventrolateral border sensory trigeminal nuclei, and in the gracile and cuneate of the DCN. In the lamprey, such glycinergic neurons are nuclei (Celio, 1990). In rats, Menétrey et al. 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' X K , h. r. . / ,?. i < : , W , \ - i - , y / Ä § Ä ^ i i ,. . i / . . ^ y / r . y . •j ; ^// r'V ;y :'íí'.^ •'. • fvÏK^' .' ‘ t > ' • •"'•■•' Ä.yyiiiipip '■ < . . . ' <■ .> :. Vy.'-'^i- •>• ï■•//rt'-. •• '•,' ' V - t e , : • * ■ ' • • y :y % % ^ V:^% > ' ' > , A ' - ■%. 't :y y^yy/y yyyv^/'" ' , \ ' : ;J ' ^ ' •' : '• ••■. ■ • y - ’m m m > ; ^ y | y i l ■ '••■•< ^ y /i", 'v > v ' w ^ ' P ^ > ; > ■■/>.:yv;:y -.'':yv;y,y/,,,>;y;-^y^.:/ Ä * » f e S 4 Ä'« //- iiiiS lliÉ I / / r / t Y '- > <■'W ¿ > ' í /:• / > • <■ - ' I '/ ■ ■ > . '.•■ -/^//y..; • '_, . S . ? -•yy^y^.y'.• y y y -^yy-'gyy •; .yyy--y ^^yyyy^;^yy:v y ' - y / ' y * . • * • X V. ' Y S < ' / < ■: ÍK#- ï ' . 'ÊiÉÉêmMlmk*•/;'/• Íí_ M r V í-Sfiy^ -y#: ■ Y ', >r / ï - •'/> , V, t. > ,.'j /,' '/ '//•■;< a . , / rv • % yyy '%v^, >;%, ' t ; f f , ' y y y y y y y / / - / / / ■ f i'¡ y fV Y ;y'<'?, >V. ••. X v. /•>. <•>. * y y , í \'/y t ■//',■ / / . ' y o:rf''.y: > '-vn'ly-v.yy^ -V /I ,■>' '•v -'X y v 7'/ V, •'..< y y y ^ : ; , . f f<. :y,, f. ''■'. -,-// y- y^.>>yyt'v ■ '/> . V : b V : ' y / ' -'¿ jjf.v //', 't. ■ Y o . ? y ’ '’Y -^y, y f > - y . - y :y y :.y ^ •• ^/- ' ^ •. .;y y y > •y''- W: 'S-.')'’ < ) ' ■ Ä MIÊKllKKÊ^ÊlÊlfUlÊÊÊÊÊtttlÎtt^ ; :v ; ^ . y „ v . V / 'ƒ • •'A >í - i '! ? . ^ • '/ / A- '•>. '/• - s ' / > . •>. )/. ^í-.l t) t ' 7A V? ■iÄ'i'i V \ THE ANURAN DORSAL COLUMN NUCLEUS 215 1993a,b). In mammals, glycinergic colls of different sizes to DCN neurons projecting to various brainstem areas have been observed in the gracile and cuneate nuclei including pretectum, superior colliculus, and pontine nu (Porucho et al., 1992). clei, related to motor processing. Even though cytourchitectonic studies do not clearly Descending control of the DCN, so prominent in mam define the anuran DON, this nucleus is characterized by its mals (see Willis and Coggeshall, 1991 for review), seems to somatotopic organization of primary afferent projections be rather restricted in anurans. After injections of tracers from the spinal cord (Antal et ah, 1980; R. usculenta; into the DCN, labeled cells were found bilaterally in the Nikundiwe et al., 1982, X. I cievis). Data in R. perezi (M. cerebellar nucleus, in the ventral nucleus of nerve VIII, and Muñoz et al., 1991) indicate a similar pattern of arrange in the reticular formation at levels between motor nuclei ment, whereby primary afferents from lumbar and thoracic VII and IX including the inferior raphe nucleus. In mam dorsal root ganglia innervate the medial, “gracile” compart mals, the transmission of sensory information through the ment of the DON, whereas those from cervical ganglia dorsal column-medical lemniscus pathway is controlled by innervate its lateral, “cuneate” compartment as well as the pathways from the cerebral cortex (e.g., Kuypers, 1958; spinal or descending trigeminal nucleus. The dorsal funicu Kuypers and Tuerck, 1964), red nucleus (Edwards, 1972; lar projection continues rostrally to innervate the vestibu Weinberg and Rustioni, 1989), vestibular nuclei (Weinberg lar nuclear complex and, rather abundantly, the granular and Rustioni, 1989), cerebellum (Sotgiu and Cesa-Bianchi, layer of the cerebellum (Antal et al., 1980; Székely et al., 1972), and reticular formation (Willcockson et al., 1987; 1980). Fibers terminating in the vestibular nuclei and in Weinberg and Rustioni, 1989). Therefore, with the possible the cerebellum arise from limb-innervating spinal ganglia exception of the red nucleus, a comparable brainstem (Antal et al., 1980; Gonzalez et al., 1984). The non-primary "control” of the DCN is found in anurans. spinal aderen ts or postsynaptic dorsal column system A major part of the present study focused on the efferent (PDCS) also appears to be arranged so mato topically. The connections of the DCN, particularly on the targets of the presence of such a PDCS has now been demonstrated medial lemniscus. The existence of a dorsal column-medial throughout terrestrial vertebrates (e.g., Rustioni, 1973; lemniscal system in amphibians remained a much debated Angaut-Potit, 1975a,b; Rustioni and Kaufman, 1977; Ben question until the early 1980s. Subsequently, Vesselkin and nett et al., 1984; Gieslor et al., 1984; Funke, .1988; ten co-workers (Vesselkin et al.} 1971; Vesselkin and Kovacevic, Donkelaar and de Boer-van Huizen, 1991; Fritz and Strit- 1973), Silvey et al. (1974), andNeary and Wilczynski (1977) zel, 1994). In mammals, the cells of origin of these non described a contralateral projection of the DCN or “perisoli- primary afferent projections to the DON, or postsynaptic tary band” (Neary and Wilczynski, 1977) to thalamic dorsal column neurons, have been shown to transmit nuclei. With electrophysio logical techniques, Urbán and nociceptive information (Uddenberg, 1968; Angaut-Petit, Székely (1982) noted slow negative potentials from the 1975b; Bennett et al., 1984; Kamogawa and Bennett, 1986), posterocentral nucleus of the thalamus in response to at least in cats. stimulation of the second dorsal root, the dorsal column, The lateral part of the anuran DON is innervated by and the DCN. In the present study the course and site of fibers from the descending tract of the trigeminal nerve, termination of the medial lemniscus was shown by antero arising from the descending part of the trigeminal, facial, grade tracing (Figs, 8,9) and its cells of origin by retrograde glossopharyngeal, and vagal nerves (Fig. 12; see also Rubin- labeling of the DCN from its main targets, i.e., the ventral son and Friedman, 1977; Mates/ and Szekoly, 1978; Fuller, thalamus, the lateral part of the torus semicircularis, and 1979; Lowe and Russell, 1982; Altman and Dawes, 1983; the cerebellar cortex (Figs. 11, 12). The use of a new and Stuesse et: al., 1984; Oka et al., 1987; Gonzalez et al., 1998; powerful anterograde tracer like BDA made it possible to M. Muñoz et al., 1994). In contrast, lateral line nerve identify even fine terminal fields and the scattered fibers in projections, present in permanently aquatic species such as the thalamus. The data obtained are summarized in Figure X. latwis, strictly avoid the DON (Lowe and Russell, 1982; 13. Since it was hardly possible to restrict tracer applica Altman and Dawes, 1983; Fritzsch et al., 1984; Will et al., tions to the DCN, in such injections the adjacent nucleus of 1985a). the solitary tract and the descending nucleus of the trigemi The most lateral part of the anuran I)CN is also inner nal nerve as well as fibers of passage (e.g., spinal primary vated by substance P- and CGRP-imnuinoreaetive fibers afferents to the cerebellum) might be involved. By retro passing via the tract of Lissauer (see also Rosenthal and grade labeling the origin of the medial lemniscal projections Cruce, 1985; Adli et al., 1988; Petkó and Santa, 1992). In was verified. It should be emphasized that the ventrolateral addition to this peptidergic primary afferent projection to part of the DCN found to project to the thalamus and the DCN, the anuran DON is innervated by Leu-Enk, NPY, particularly to the torus extends cai; dally as far as the and 5-HT-im mu no reactive libers in line with data by Ueda second spinal segment. The dendrites of these cells are et ah (1984), Merchenthaler et al. (1.989), and Lazar et al. mainly directed to the dorsolateral funiculus, whereas their (1990). This serotonergic and peptidergic innervation of the axons join the contralateral medial lemniscus. A certain DCN is in line with immunohistochemical data in mammals similarity to the mammalian lateral cervical nucleus, known (e.g., Steinbusch, 1981; Westman et ah, 1984; Halliday et to receive somatosensory information via the spinocervical al., 1988; Ihuki et al., 1989; Tamatani et alM 1989; Conti et tract and projecting contralaterally via the medial lemnis al., 1990; Fabri and Conti, 1990; Blomqvist and Broman, cus (Willis and Coggeshall, 1991), seems likely. 1993). Since after tracer applications to the DCN retro- The medial lemniscus could be traced throughout the gradely labeled neurons were observed in the (serotonergic— brainstem and into the diencephalon. Along its course, the see Ueda et al., 1984) raphe nucleus, it seems likely that medial lemniscus gives off collaterals to various parts of this nucleus is the source of the serotonergic innervation of the reticular formation, to the octavolateral area, and to the the DCN. In rats, Willcockson et al. (1987) observed granular layer of the cerebellum. At mesencephalic levels, serotonergic terminals in apposition to neurons of the DCN the medial lemniscus primarily innervates the lateral part that project to the thalamus, whereas in cats and monkeys, of the torus semicircularis (also noted by Coiner and Blomqvist and Broman (1993) observed serotonergic input Grobstein, 1981; Wilczynski, 1981; Neary and Wilczynski, 216 A. MUÑOZ ET AL. 1986; Neary, 1988) and the anterodorsal and anteroventral tegmental nuclei as well as the red nucleus and the interstitial nucLeus of the fasciculus longitudinalis medialis. Whereas in R. perezi medial lemniscal fibers do not reach the tectum mesencephali, in X. laevis intermediate and deep tectal layers are innervated in agreement with retro grade tracer data (Wilczynski and Northcutt, 1977; Zittlau et al., 1988; Hofmann et al., 1990; Masino and Grobstein, 1990). Beyond the midbrain, both dorsal and ventral tha lamic areas are innervated by the medial lemniscus. The ventral parts of the posterior and central nuclei of the dorsal thalamus are reached by a few thin, varicose, fibers, but the ventromedial thalamic nucleus and the nucleus of the posterior tubercle are far more densely innervated. In two cases in R. perezi, a few fibers also reached the anterior nucleus of the dorsal thalamus. No projections beyond the diencephalon were observed. Extralemniscal projections were found to the ipsilateral cerebellar cortex, confirming retrograde tracer data (González et al., 1984), and bilaterally to the spinal cord. The ipsilateral spinal projection from the DCN was previously observed inX. laevis (ten Donkelaar et al., 1981), Hence, the present study not only further substantiated the presence of a rather well-developed dorsal column- medial lemniscus system in anurans, but also showed that its mesencephalic and diencephalic targets are much more extensive and diverse than suggested in previous studies (Vesselkin et al., 1971; Silvey et al., 1974; Neary and Wilczynski, 1977; Comer and Grobstein, 1981; Wilczynski, 1981; Forehand and Farel, 1982; Urbán and Székely, 1982; Neary, 1988). The anuran “lemniscal pathway” appears to be basically similar to that of amniotes (reptiles: Ebbesson, 1978; Siemen and Kiinzle, 1994a; birds: Wild, 1989; mam mals; e.g., Hazlett et al., 1972; Hand and van Winkle, 1977; Feldman and Kruger, 1980; Berkley et al., 1986; see also Willis and Coggeshall, 1991 for a summary of mammalian studies), although in mammals the widespread thalamic projections should be particularly emphasized. In the red eared turtle, Pseudemys scripta elegans, Siemen and Kiinzle (1994a,b) noted a direct ascending projection from the most medial part of the DCN area, by-passing the thalamus, to the basal part of the telencephalon. At first sight, the mesencephalic target of the anuran medial lemniscus seems to be quite different from the amniote mesencephalic target. For the opossum, RoBards et al. (1976) introduced the term intercollicular terminal zone for the common target of projections from the dorsal column nuclei, spinal cord, and sensorimotor cortex in the central midbrain. In reptiles, Ebbesson (1967, 1969) intro duced the term intercollicular nucleus for the mesence phalic target of ascending spinal projections. In this nucleus, a projection from the dorsal column nucleus terminates as well (Ebbesson, 1978; see also Belekhova et al., 1985; Pritz and Stritzel, 1989b), It seems likely that this intercollicular zone, nucleus, or “midbrain somatosensory area” (Pritz and Stritzel, 1989b), characterized by at least an input from the spinal cord and DCN, is a major integrative center of the somatosensory system. Pritz and Stritzel (1990) showed that the medialis complex in the dorsal thalamus is the thalamic target of the midbrain somatosensory intercollicu lar area. In anurans, the main midbrain target of the medial lemniscus is formed by the lateral part of the torus semicir- cularis (Comer and Grobstein, 1981; Wilczynski, 1981; Neary and Wilczynski, 1986; Neary, 1988; the present study). The anuran torus semicircularis is a major integrat Fig. 13. Diagram summarizing the fiber connections of the anuran ing center for a number of sensory and non-sensory affer- dorsal column nucleus shown in a dorsal view of the brain of R. perezi. THE anurán d o r sa l co lum n n u c l eu s 217 ents in addition to its auditory input and may well serve a fore, somatosensory information could reach the medial role similar to the one the tectum meseneephali serves for pallium via multisynaptic routes. the visual system (Wilczynski and Capranica, 1984). It Another telencephalic structure in which somatosensory includes a laminar nucleus, a principal nucleus, a magnocel- activity was recorded is the striatum (Vesselkin et al., 1971; lular nucleus, and two smaller nuclei (Potter, 1965), each of Vesselkin and Kovacevic, 1973). The anuran striatum which receives a particular set of afferents (e.g., Wilczynski, receives a major thalamotelencephalic input from nuclei 1988; Feng and Lin, 1991). Physiological studies (Comer relaying sensory information from the midbrain roof, from and Grobstein, 1981) in R. pipiem suggest a certain overlap the torus semicircularis, and from ventral diencephalic of tactile and auditory information: the very dorsolateral structures, receiving spinal and DCN-medial lemniscal torus is almost exclusively concerned with tactile informa afferents (Scalia and Colman, 1975; Vesselkin et al., 1980; tion; auditory activity is most often found to be localized in Wilczynski and Northcutt, 1983a; Lázár and Kozicz, 1990), central parts of the torus, but in between the two, multimo The striatum in amphibians appears to be able to influence dal (tactile and auditory) activity is found. Toral afferents the midbrain roof via the p re tec turn and various midbrain also arrive from the vestibular (Wilczynski, 1981), and, in and isthmal nuclei (Wilczynski and Northcutt, 1983b). The X. laevis, from the lateral line system (Will et ah, 1985b; anuran striatum plays a crucial role in processing sensory Lowe, 1986; Zittlau et ah, 1986). The laminar toral nucleus information as well as in coordinating all telencephalic not only receives DON efferent« but also spinal (Ebbesson, output to lower brainstem motor centers. Both visual 1976; A. Munoz et aL, in preparation) and trigeminal (Gruberg and Ambros, 1974) and auditory (Mudry and afferents (Comer and Grobstein, 1981; M. Munoz et al., Capranica, 1980) activity was recorded from the striatum. 1994), and so—at least partly—represents a midbrain Further, electrical stimulation of the sciatic nerve evoked somatosensory area. The multimodal laminar nucleus as potentials in the striatum (Vesselkin et al., 1971; Vesselkin well as the mainly auditory magnocellular nucleus exten and Kovacevic, 1973). The sensoi’y input to the striatum is sively innervate the central and posterior nuclei (Frontera’s relayed in the anterior division of the lateral thalamic nucleus posterocentralis; see Frontera, 1952) of the dorsal nucleus (visual information: Lázár, 1969; Scalia, 1976), in thalamus, whereas the ascending projections of the princi the central thalamic nucleus (auditory and also somatosen pal nucleus are restricted to the caudal part of the posterior sory information: Hall and Feng, 1987; Feng and Lin, 1991; thalamic nucleus (Hall and Feng, 1987; Feng and Lin, present study), and in the ventromedial thalamic nucleus 1991). The laminar nucleus also innervates the ventrome (somatosensory information: Neary and Wilczynski, 1977; dial thalamic nucleus (Feng and Lin, 1991; A. Munoz and present study). In R. perezi, tracer applications to the ten Donkelaar, unpublished observations), i.e., the main striatum showed a direct striatal projection arising from diencephalic target of the medial lemniscus {present study) cells in the lateral aspect of the ventromedial thalamic as well as of the spinothalamic tract (A. Munoz et al., 1994), nucleus (A. Muñoz, unpublished observations), in line with The central thalamic nucleus extensively projects to the observations by Vesselkin et al. (1980) as well as by Lázár ipsilateral striatum (Vesselkin et al., 1980; Wilczynski and and Kozicz (1990). Vesselkin et aL (1980) also noted a direct Northcutt, 1983a; Neary, 1988). Hence, this DCN-torus- striatal projection from the torus semicircularis, whereas central thalamic nucleus-striatal pathway is one way by Lázár and Kozicz (1990) found a few faintly labeled small cells in the nucleus of the posterior tubercle projecting to which somatosensory information may reach the telencepha lon. the lateral wall of the telencephalon including the striatum (see also Wilczynski and Northcutt, 1983a). Somatosensory Although the anuran dorsal column-medial lemniscus information to the striatum may thus be relayed via the system is basically similar to that of amniotes, large dorsal thalamus (the central thalamic nucleus), the ventral differences are found with regard to the telencephalic thalamus (the ventromedial thalamic nucleus), and the targets of this pathway. Therefore, a few remarks on the nucleus of the posterior tubercle, a separate diencephalic telencephalic structures receiving somatosensory informa region (Neary and Northcutt, 1983). Which thalamic nuclei tion in anurans seem appropriate. Physiological studies really relay somatosensory information to the striatum revealed somatosensory activity within the medial pallium (and medial pallium) is now being studied in a series of (e.g., Supin and Ousel*nikov, 1964; Karamian et al,, 1966; double-labeling experiments. Northcutt, 1970; Vesselkin and Kovacevic, 1973), possibly relayed in the dorsal thalamus. Since the anterior thalamic nucleus is the only thalamic nucleus innervating the medial ACKNOWLEDGMENTS pallium (Scalia and Colman, 1975; Vesselkin and Erma kova, 1978; Kicliter, 1979; Neary, 1984; Northcutt and This research was supported by an ENP short-term Ronan, 1992), somatosensory information to this pro fellowship of the European Science Foundation to A. Mu nounced telencephalic structure, also known as the archipal- ñoz, a NATO Collaborative Research Grant (930542) to liuni (Ariens Kappers et al., 1936; Clairambault and Derer, H.J. ten Donkelaar, and by a grant from the Spanish 1968) or the primordium hippocampi (Herrick, 1910; Hoff Government (DGICYT PB 93-0083) to A. González. The man, 1963), must relay in the anterior nucleus. However, authors thank Mrs. Roelie de Boer-van Huizen and Mrs. since spinal afferents to the anterior thalamic nucleus' Ine Bergervoet-Vernooy for excellent technical assistance, either via the spinothalamic tract (A, Munoz et al., 1994) or Mrs. Marlu Ackermans for making the final versions of the via the dorsal column-medial lemniscal pathway (Neary drawings, and Ms. Inge Eijkhout for secretarial assistance. and Wilczynski, 1977; pi'esent study)—are rather limited, The valuable comments of Dr. William L.R. Cruce are alternative routes must be available, possibly via the poste gratefully acknowledged. rior thalamic nucleus, known to project to the anterior thalamic nucleus (Neary and Wilczynski, 1979; see also Neary, 1990; Northcutt and Ronan, 1992), It should also be LITERATURE CITED noted that the dendrites of cells in the anterior thalamic Adams, J.C. ( 1981) Heavy metal intensification of DAB-based HRP reaction nucleus penetrate the central nucleus (Neary, 1990). There product. J. Histochum. Cy toe hem. 29:115. 218 A. MUÑOZ ET AL. Adli, D.S.H., B.M. Rosenthal, G.L. Yuen, R.H. Ho, and W.L.R. Cruce (1988) Ebbesson, S.O.E. (1969) Brain stem afferents from the spinal cord in a sample of Immunohistochemical localization of substance P, somatostatin, enkepha reptilian and amphibian species. Ann. N.Y. Acad. Sci. 767:80—101. lin* and serotonin In the spinal cord of the Northern leopard frog, Rana Ebbesson, S.O.E. (1976) Morphology of the spinal cord. In R. Llinás and W. pipiens. J, Comp. 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