Distribution morphology and central projections of mesencephalic trigeminal neurons in the frog Rana ridibunda.код для вставкиСкачать
THE ANATOMICAL RECORD 235:165-177 (1993) Distribution, Morphology, and Central Projections of Mesencephalic Trigeminal Neurons in the Frog Rana ridibunda MARGARITA MUNOZ, ALBERT0 MUNOZ, AND AGUSTfN GONZALEZ Departamento de Biologia Celular, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Spain ABSTRACT The distribution, morphology, And central projections of the mesencephalic trigeminal neurons in the frog R a m ridibunda were studied with tracing techniques. Retrograde tracing with horseradish peroxidase (HRP) or the fluorescent tracer Fluorogold, and anterograde tracing by means of Phuseolus uulgaris leucoagglutinin, the fluorescent dye DiI, and HRP were used. The mesencephalic trigeminal nucleus (MesV) of Rana ridibunda is formed by a population of 100 to 125 unipolar or multipolar cells that are scattered on both sides of the rostra1 mesencephalic tectum. Subpopulations of Mes V cells were labeled after tracer application to ophthalmic, maxillary, and mandibular trigeminal branches, separately. Differences in the morphology and distribution of cells in these experiments were not evident but the number of neurons labeled via the maxillary nerve was always the highest. Mes V cells have a single central branch that courses caudally in the brainstem. At different levels, it bifurcates into a peripheral branch, which leaves the brain via the trigeminal root, and a descending branch, which terminates in a region in, or close to, the trigeminal motor nucleus and in a supratrigeminal location. The lack of a distinct somatotopy in the distribution of Mes V cells and the lack of projections caudal to the trigeminal motor nucleus as revealed in this study with a wide variety of tracers are in striking contrast to previous data provided for other amphibians. o 1993 Wiley-Liss, Inc. Key words: Anuran, Brainstem, Trigeminal nerve, Mesencephalic trigeminal nucleus, Axonal transport, HRP, PHA-L, DiI The mesencephalic nucleus of the trigeminal nerve (Mes V) is a conspicuous cell group formed by large neurons that stain well with classical techniques. Thus, its presence in the brain was confirmed in early studies in all jawed vertebrates (Johnston, 1909; Weinberg, 1928; Woodburne, 1936). Mes V neurons have been compared to primary sensory ganglion cells with peripheral processes running in the trigeminal nerve but with cell bodies included in the CNS. The peripheral processes carry somatosensory information centripetally from proprioceptors in the head region. With some variations depending on the species, Mes V fibers reach, via the trigeminal nerve, muscles of the jaw and oral region, teeth, and extraocular muscles (AlvaradoMallart et al., 1975; Darian-Smith, 1973; Wild and Zeigler, 1980; Matsez 1981; Jacquin et al., 1983; Capra et al., 1985; Shigenaga et al., 1988a-c). In several species, it has been shown that each trigeminal subdivision (ophthalmic V1, maxillary V2, and mandibular V3) contains Mes V fibers (Dacey, 1982; Jacquin et al., 1983; Ryu et al., 1983; Barbas-Henry and Lohman, 1986; Puzdrowski? 1988). Once in the brainstem, the peripheral branches bifurcate giving rise to central and descending branches. While the central branches reach their parent Mes V cell bodies, the descending branches turn caudally in the brainstem. Studies in0 1993 WILEY-LISS, INC. vestigating the terminal fields of the descending branches have produced controversial results and have suggested the presence of important species differences (Darian-Smith, 1973; Matesz, 1981; Matsushita et al., 1981; Arends and Dubbeldam, 1982; Dacey, 1982; Hiscock and Straznicky, 1982; Luschei, 1987). Targets for these central projections include the trigeminal motor, principal sensory and descending nuclei, supratigeminal nucleus, oculomotor nuclei, branchiomotor column, cerebellum, and spinal cord. The Mes V nucleus has been studied with experimental techniques in various vertebrates. Together with the classically described unipolar cell bodies, multipolar cells have also been found in this cell group (MacDonnell, 1980; Lowe and Russell, 1984; Walberg, 1984; Faccioli et al., 1985; Shigenaga et al., 1988a,b). In addition, electron microscopic observations demonstrated the presence of synaptic terminals on Mes V neurons (Hinrichsen and Larramendi, 1970; Lucchi et al., 1972; Alley, 1973; Witkovsky and Roberts, 1976; Munoz and Gonzalez, 1990). This would suggest an integrative Received February 3, 1992; accepted April 28, 1992. Address reprint requests to Margarita Munoz, Ph.D., Departamento de Biologia Celular, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Spain. 166 M. MUNOZ ET AL. function for the Mes V in addition to its involvement in the monosynaptic reflex arc producing jaw closure, demonstrated earlier for several vertebrates (Corbin and Harrison, 1940; Manni et al., 1965; Azzena and Palmieri, 1967; Roberts and Witkovsky, 1975). Recently, several studies have dealt with the trigeminal system in diverse amphibians including both urodeles (Gonzalez and Munoz, 1988b;Roth et al., 1990) and anurans (Rubinson, 1970; Matesz and Szekely, 1978; Lewis and Straznicky, 1979; Hiscock and Straznicky, 1982; Lowe and Russell, 1984). Regarding the Mes V, there are differences of opinion in these studies, mainly due to the technical approaches used. As part of our research on the trigeminal system of amphibians, the distribution, morphology, and projections of the Mes V have been studied in the frog Rana ridibunda. The data are based upon experiments with modern tract tracing techniques with retrogradely transported tracers applied to the peripheral trigeminal branches and anterogradely transported tracers injected or positioned into the area of the mesencephalic tectum where Mes V cells were observed. MATERIALS AND METHODS This report is based on 85 adult specimens of Rana ridibunda. In 47 animals, horseradish peroxidase (HRP) or the fluorescent tracer Fluorogold was applied to the trigeminal root or one of the three subdivisions of the trigeminal nerve. All approaches were unilateral. The animals were anesthetized by being placed in a 0.3% solution of tricaine methanesulphonate (MS 222, Sandoz). In 32 animals HRP was applied to the trigeminal root (n = 6), the ophthalmic nerve (n = €9, the maxillary nerve (n = 101, and the mandibular nerve (n = 8). In some experiments 50% HRP (type 1, Boehringer) dissolved in 0.9% saline was delivered through small plastic tubes a t the central ends of the transected nerves; in other experiments detergentsoaked HRP chips (Nonidet P-40, Sigma N6507, St. Louis, MO) were implanted in the trigeminal root or in one of the three nerves. Following survival times of 7-15 days, the animals were reanesthetized and perfused transcardially with saline, followed by a mixture of 1.5% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The brain and spinal cord were removed and further fixed in the perfusion mixture for 1-4 hours. They were then immersed in a mixture of phosphate buffer and 30% sucrose solution for 3-5 hours a t 4"C, subsequently embedded in a solution of 15% gelatin with 30% sucrose added, and stored overnight in a 4% formaldehyde solution a t room temperature. Sections were cut a t 40 pm thickness in the transverse plane on a freezing microtome. Alternate sections were processed according to a slightly modified procedure (Mesulam, 1978) with tetramethylbenzidine as chromogen and the Adams' (1981) heavy metal intensification of diaminobenzidine-based reaction product. The sections were counterstained with neutral red or cresyl violet. In 9 animals a 2% solution of Fluorogold was applied to the severed nerves following a similar surgical procedure a s described for the 50% HRP experiments. The animals were allowed to survive 6-9 days and were then perfused with 4% paraformaldehyde. After removal from the skull, the brains were embedded in 15% gelatin with 30% sucrose, and frontal sections of 40 pm thickness were cut on a freezing microtome. All sections were mounted immediately from a 0.2% gelatin solution and were examined with a n epifluorescence microscope (Zeiss). In a second series of experiments the anterograde tracer Phaseolus uulgaris leucoagglutinin (PHA-L, n = 10) or HRP was injected into the tectum in order to study the projections of the Mes V cells. Similary, HRP was injected into the medulla (n = 13) and spinal cord (n = 6) to study the distribution of retrogradely labeled cell bodies in the tectum. All injections were made iontophoretically by applying 5-8 pA positive pulsed current (7 s on17 s off) to the tracer solution (10%HRP, in most cases with 10% Saponin or Nonidet P-40; 2.5% PHA-L) in a glass micropipete (outer tip diameter 1015 pm) for a period of 15-30 min. The animals survived for 6-10 days. They were then reanesthetized and perfused transcardially with saline followed by a mixture of 1% paraformaldehyde and 2.5% glutaraldehyde (for PHA-L experiments)or 1% paraformaldehyde and 1.25% glutaraldehyde (for HRP experiments) in 0.1 M phosphate buffer, ph 7.4. After posfixation for 2-4 hr, the brains were stored overnight a t 4°C in 30% phosphate-buffered sucrose. Subsequently, they were embedded in gelatin, as previously described, and cut a t 40 pm on a freezing microtome. The protocol for the immunostaining of PHA-L in amphibian brains followed that previously described for reptiles (Russchen et al., 1987). The HRP was visualized as in the experiments where it was peripherally applied. The fluorescent dye DiI (l,la,dioctadecyl-3,3,3a,3atetramethylindolcarbocinine perchlorate; Molecular Probes, Inc., Eugene, OR) was used in the last series of experiments (n = 5). Its antrograde diffusion and staining was observed in fixed tissue as described by Godement et al. (1987). Thus, crystals of the dye were positioned in different, selected tectal regions of brains that were previously fixed in 4% paraformaldehyde (pH 7.4) for at least 5 hr. Tissue was stored following the placement for 6-9 weeks in the same fixative and kept in the dark at room temperature. The brains were then cut in the transverse plane a t 60-80 pm thickness with a vibratome. The sections were collected in serial order in buffer and, for observation, were mounted onto glass slides. All preparations were observed with a n epif luorescence microscope (Zeiss) equipped with a rhodamine filter set for viewing the orange-red DiI fluorescence. RESULTS Peripheral Course of the Trigeminal Nerve In Rana ridibunda the trigeminal nerve root emerges from the ventrolateral aspect of the rostral rhombencephalon, just caudal to the cerebellum (Fig. 1). Shortly after, it reaches the trigeminal ganglion, a n enlargement attached to the rostral wall of the otic capsule. Three nerve trunks originate from this ganglion: the facial nerve, whose sensory fibers have their cell bodies in the caudoventral portion of the ganglion, and the two main subdivisions of the trigeminus, i.e., the ophthalmic nerve (Vl) and the mixed maxillo-mandibular nerve (V2 V3). The ophthalmic nerve originates from the rostral part of the trigeminal ganglion and courses toward a + TRIGEMINAL MESENCEPHALIC NUCLEUS O F THE FROG 167 Fig. 1. Schematic drawing indicating the distribution of the peripheral branches of the trigeminal nerve. The black bars indicate the sites of application of retrograde tracers to the separate ophthalmic (Vl), maxillary (V2), and mandibular (V3) trigeminal branches. Scale bar indicates 0.5 cm. Abbreviations A ASP Cb D Dh FP IP Is Max MesV Mm articular bone angulosplenial bone cerebellum dentary bone dorsal horn frontaparietal bone nucleus interpeduncularis nucleus isthmi maxillary bone nucleus mesencephalicus nervi trigemini mentomeckelian bone N nasal bone Pm premaxillary bone PrV nucleus princeps nervi trigemini quadrate bone Q quadrotojugal bone &i Ri nucleus reticularis inferior Rm nucleus reticularis medius Rs nucleus reticularis superior S squamosal bone Sol tractus solitarius Tect tectum mesencephali Tor torus semicircularis vo ventriculus opticus I11 nucleus nervi oculomotorii IV nucleus nervi trochlearis v1, v 2 , V3 ophthalmic, maxillary, and mandibular nerves V root trigeminal root Vd nucleus descending nervi trigemini Vm nucleus motorius nervi trigemini VII nervus facialis VIII nuclei nervi octavi Xm nucleus motorius nervi vagi subcutaneous position at the level of the premaxilla. In its course, it parallels the frontoparietal bone and lies dorsal to the superior rectus muscle. Fine sensory branches to the extraocular muscles in the orbit are given off a t various points. The maxillo-mandibular nerve emerges from the lateral aspect of the trigeminal ganglion. This thick nerve trunk runs laterally and passes between the eyeball and the eardrum. It then splits into separate maxillary 072) and mandibular (V3) nerves. V2 bends rostrally and borders the orbit lateroventrally toward the upper mandible. In its way it sends numerous sensory branches t o the extraocular muscles. In turn, V3 courses ventrally, close to the squamosal and quadrate bones, and passes into the inferior mandible. Distribution and Morphology of Mes V Neurons The localization of the Mes V cells was studied by means of retrograde transport of HRP or the fluorescent tracer Fluorogold. The tracers were unilaterally applied to either the trigeminal root or individual trigeminal nerves, V1, V2, and V3 (see Fig. 1). Experiments with HRP applied to the single trigeminal root resulted in labeling of all trigeminal components, since this tracer is transported both retrogradely and anterogradely. Thus, the motor nucleus and the primary afferent projections were clearly stained together with the Mes V. When the fluorescent tracer 168 M. MUNOZ ET AL. was used, the Mes V neurons and the trigeminal motoneurons were retrogradely labeled, and anterograde labeling of afferent fibers was almost absent. Mes V cell bodies were always labeled in the ipsilatera1 mesencephalic tectum where they form a scattered population (Fig. 2A-C). Rostrocaudally, they extend from the anterior tectal pole (Figs. 2A,B, 3a) to the level of the trochlear nucleus (Figs. 2C, 3b). Within the tectum, they are mainly located in layers 2 and 4.However, a few neurons are present in layer 6 (Fig. 4a). Only occasionally one or two cells were observed in the ependymal layer (Figs. 3b, 4b) or within the ventricle itself. An average of 100-125 Mes V cells were labeled in each experiment with tracer application to the trigeminal root. The density of cell distribution in the tectum is not uniform and the highest population is located a t rostral levels, medial to the rostral tip of the tectal ventricle (Figs. 2B, 3a). In this position, the generally dispersed Mes V neurons form a rather compact group with several adjacent cells. Mes V cell bodies are always large and most often present a globe-to-ovoid profile (20 x 30 km in diameter). They are characteristically unipolar with a single process that emerges dorsally, crosses several tectal layers, and leaves the tectum (Figs. 3a,b, 4a,b). However, a small population (7-15%) are multipolar with 4 to 7 somatic processes (Fig. 3a,b). In a second set of experiments, the isolated trigeminal nerves V1, V2, and V3 were exposed to the tracers. All three nerves carry peripheral branches of the Mes V neurons, and labeled somata were always present in the ipsilateral tectum. No differences in cell morphology were observed after differential labeling of trigeminal nerves, and thus, unipolar and multipolar neurons were constantly observed. The analysis of the distribution of labeled cell bodies failed to reveal clear differences among individual nerves. The distribution of the somata always followed the same pattern and this coincided with t h a t described for the experiments in the trigeminal root. Thus, a somatotopic arrangement in the mesencephalic tectum is hard to recognize. However, experiments in V3 labeled the lowest population in the caudal portion of the Mes V. The number of labeled cells varied widely not only between nerves, but among experiments with treatments of the same nerve. Cells labeled via V2 ranged from 39 to 63 while the labeled populations via V1 and V3 ranged from 14 to 35, and from 18to 39, respectively. Organization of Mes V Cell Processes In order to study the organization of the processes arising in the Mes V neurons, experiments with the retrograde and anterogradely transported HRP were analyzed. When the tracer was applied to the trigeminal root or its peripheral branches, a contingent of coarse fibers was identified in the dorsal part of the Vth nerve root (Fig. 2F). These fibers constitute the peripheral branches of the Mes V processes. Once in the rhombencephalon, they turn rostrally and form a n ascending tract of loose coarse fibers (Figs. 2E, 5a). At isthmic levels, the fibers separate into a major component that borders the isthmic nucleus laterally and a minor component that crosses it or moves medially into a periventricular position (Figs. 2D, 5b). They pass through the torus semicircularis and rejoin beneath the tectum (Figs. 2C,D, 5b). They then bend dorsally and enter layer 7 of the tectum (Fig. 5b), finally reaching their parent cell bodies. At their entrance in the rhombencephalon, some fibers bifurcate giving off thinner fibers that are oriented medially and somewhat caudally in the brainstem. Similarly, fine fibers were observed along the course of the ascending tract. This set of fine branches forms the descending component of the Mes V cell processes. Thus, from the point of bifurcation and up to the Mes V cell bodies the coarse fibers represent the central branches of the Mes V axons. Thereby, the single process of a Mes V neuron is made up of peripheral, descending, and central branches. Their respective lengths vary considerably depending on the location of the bifurcation point. In the experiments with HRP, descending branches of the Mes V cells reach areas in, and close to, the trigeminal motor nucleus (Figs. 2E,F, 5a). However, it was not possible to establish their actual place of termination and their caudalmost extent since HRP also labeled the primary sensory projections and the motor neurons in the trigeminal nucleus (Figs. 2F, 5a). Therefore, different categories of fibers are intermingled and tracing of a single trigeminal component was not possible. Central Projections of the Mes V Neurons To investigate the course and termination sites of the descending branches of the Mes V cells, anterogradely transported tracers (PHA-L, HRP, DiI) were applied to the areas in the tectum where the highest population of Mes V neurons are located (Figs. 6A, 7a, 8a). Adjacents sections were stained with cresyl violet and used as controls to visualize the high number of Mes V cells in the injection sites. The results obtained with each tracer were consistent with each other. However, depending on the tracer, the application site, the course of peripheral branches, and the termination of descending branches were distinctly observed. The combination of the three substances used gives a complete picture of the projections (see the technical considerations below). Coarse fibers were directed caudally and ventrally from the injection site, following a pathway similar to that observed for the ascending Mes V tract (Fig. 6A-C). These fibers were clearly distinguished from those, more laterally located, of the tectobulbar and tectospinal pathways. At different levels in their path and up to a level immediately rostral to the trigeminal nerve root (Fig. 6D), the fibers arborize, giving off collaterals that distribute in the direction of the trigeminal motor nucleus. The rostral half of the motor nucleus in particular is reached and its ventrolateral aspect is richly innervated (Fig. 6D,E). I n addition, a second contingent of thin fibers reaches a detached area of medium-sized neurons dorsal and lateral to the motor nucleus of the trigeminus (Figs. 6D, 7b, 8b). It was not possible to follow the descending branches of the Mes V further caudally than the level of the trigeminal motor nucleus. In order to confirm the projection from the Mes V, HRP injections were iontophoretically placed in the regions of the trigeminal motor nucleus and lateral and dorsal to it (Fig. 9A). Only those injections that clearly avoided the area of the ascending Mes V tract were TRIGEMINAL MESENCEPHALIC NUCLEUS OF THE FROG d 5 1 1 II II A+ F - Fig. 2. Distribution of labeled neurons and fibers in the mesencephalon and rostra1 rhombencephalon after HRP application to the trigeminal root. The appropriate level of the sections A-F is indicated in the schematic drawing above. Scale bar represents 1 mm. 169 170 M. MUNOZ ET AL. Fig. 3. Photomicrographs showing retrogradely labeled MesV neurons after HRP application to the trigeminal root. Labeled cells are located at the level comparable to that of Figure 2B (a)and 2C (b).Scale bars indicate 100 em. Fig. 4. Retrogradely labeled Mes V cells in the mesencephalic tectum after fluorogold (a)or HRP (b) application to the trigeminal root. Note their variable morphology and location within the tectal layers. Scale bars represent 20 km in (a) and 40 km in (b). considered, and, in all of these experiments, Mes V cells were retrogradely labeled in the ipsilateral tecturn. In the literature it has been suggested that the Mes V receives input from the sensory nuclei of the trigeminal nerve, the branchiomotor nuclei a t obex levels, TRIGEMINAL MESENCEPHALIC NUCLEUS OF THE FROG 171 Fig. 5. a: Cross section through the rostral part of the rhombencephalon showing retrogradely labeled motoneurons and anterogradely labeled primary afferents following HRP application to the trigeminal root. Arrow points to the labeled fibers of the mesencephalic trigem- inal tract. b: Same experiment as in a where the labeled fibers of the mesencephalic trigeminal tract are shown at more rostral mesencephalic levels entering ventrally into the tectum (arrow). Scale bars indicate 140 Km. and the spinal cord (see “Discussion” section). Therefore, we placed HRP injections in these structures (Fig. 9B-E) in order to determine if they are reached by the caudal extent of the descending Mes V branches. However, our results were always negative and no labeling was ever observed in the Mes V, either a t rostral or caudal levels. A summary diagram of the organization of the Mes V neuron processes found in the present study is shown in Figure 10. Only two places of termination for the descending branches are considered, i.e., the supratrigeminal region and the trigeminal motor nucleus, a s observed in our experiments. cells was comparable and the lack of somatotopical arrangement of Mes V neruons was always corroborated, independently of the tracer used. When applying HRP to the trigeminal root or ganglion, together with the retrograde transport, anterograde transport of the enzyme also occurs. Thus, the primary afferents are also labeled. This fact makes it difficult to study the descending projections from the Mes V since they probably would be intimately related with the primary sensory afferents, as proposed by Matesz and Szekely (1978) using a similar approach with the cobalt chloride technique. In order to solve this problem, sensitive anterogradely transported tracers were applied to those places in the tectum where most of the Mes V cells were observed. Iontophoretical injections of HRP and PHA-L were used. Similar results were observed with both tracers, although PHA-L allowed us to get smaller and better situated injection sites and the projections were easier to trace. Following the newly described method of staining neurons and their processes with DiI in fixed tissue (Godement et al., 1987) we applied this tracer to selected tectal regions. This method has been recently shown to be very sensitive for study of descending projections in the amphibian brain (Tan and Miletic, 1990). Although the DiI crystals are very small (15-20 pm in diameter) i t seems that a large application site is actually involved in the diffusion of the tracer (see Fig. 8a). We have observed clearly DiI labeled fibers and terminals in the same areas as found with PHA-L but also other prominent tectal efferent systems (such as the tectoisthmal and tectobulbar tracts) were always labeled. The analysis of the experiments with tectal application of anterograde tracers resulted in the lack of caudal projections that would reach the spinal cord in its dorsal horn, a s proposed with the cobalt technique in Rana esculenta (Matesz and Szekely, 1978). To corrob- DISCUSSION Technical Considerations The aim of the present study is to provide a detailed description of the distribution, morphology, and central projections of the mesencephalic trigeminal neurons in the frog Rana ridibunda by means of tract tracing techniques. From data of our previous experiments (Gonzalez and Munoz, 1988a) two negative results were noteworthy: 1)the lack of distinct somatotopy in the distribution of Mes V cells, and 2) the lack of projections caudal to the motor V nucleus. For this reason a wider variety of tracers and approaches has been used in the present study. The distribution of retrogradely labeled Mes V cell bodies was first achieved by means of HRP. However, we also used the retrogradely transported fluorescent dye Fluorogold which is also very suitable to demonstrate cell distribution in the amphibian trigeminal system (Gonzalez and Munoz, 1988b). The application procedure in the case of fluorescent substances is more tedious since they have to be used a s solutions, differently from the HRP crystals that are easier to handle. However, these methods applied in this study gave essentially identical results. Thus, the number of Mes V 172 M. MUNOZ ET AL. I I1 II A- E Fig. 6. Cross sections through the mesencephalon and rostral rhombencephalon showing the distribution of labeled fibers after PHA-L injection into the rostral mesecephalic tectum. The injection site is represented by the shaded area in level A. Scale bar represents 1 mm. orate the latter finding we injected HRP in medullary and spinal cord regions. However, even in experiments loading the upper cervical cord segments or the caudal rhombencephalon, no retrogradely labeled Mes V cells were observed although other groups located further rostral (for instance in the diencephalon) were successfully labeled. Since this result is in concordance with all similar studies of anuran amphibians (Ten Donkelaar et al., 1981; Toth et al., 1985) we do not think that our negative staining is due to a technical failure. TRIGEMINAL MESENCEPHALIC NUCLEUS OF THE FROG 173 Fig. 7. Photomicrographs of a experiment where PHA-L was injected at rostral levels of the mesencephalic tectum, just medial to the rostral aspect of the optic ventricle (a).In b labeled fibers with varicosities can be observed in the area of the trigeminal motor nucleus. Scale bars indicate 145 pm in a and 50 pm in b. Fig. 8. Photomicrographs of an experiment where a small crystal of DiI was applied to the tectum at medial rostrocaudal levels. a: Panoramic showing the distribution of labeled fibers leaving the tectum at caudal tectal levels. b: labeled fibers dorsal to the trigeminal motor nucleus (comparable to Fig. 7b). Scale bars represent 120 pm in a and 40 p m in b. The Mesencephalic Trigeminal Nucleus mesencephalon, in the rhombencephalon near the trigeminal motor nucleus (Weinberg, 1928; Ruggiero et al., 1982). In Rana ridibunda, a s in other anurans (Lowe and Russell, 1984; Kollros and McMurray, 1955; Matesz and Szekely, 1978), Mes V neurons distribute mainly over the rostral part of the tectum and, principally, in layers 2 and 4 (as distinguished by Lazar et al., 19831, with fewer cells in layer 6, the ependymal layers, and within the ventricle itself. This contrasts with the situation in urodele amphibians in which Mes V neurons are more evenly distributed in the tectum All Mes V cells labeled in Rana ridibunda by means of retrograde transport of HRP or fluorescent tracers were located in the tectum and always ipsilateral to the side of application. This general location is shared by most species studied. Only occasionally, a few contralateral Mes V cells have been reported in various species (Lowe and Russell, 1984; New and Northcutt, 1984; Gonzalez and Munoz, 1988b). In addition, in mammals, some Mes V cells are located outside the 174 M. MUNOZ ET AL. Ill I I A- E Fig. 9. Schematic drawings of transverse sections through the rhobencephalon (A-D) and cervical spinal cord (El. Several injection sites where retrograde tracers were located are represented by shaded areas. Scale bar indicates 1mm. from rostral levels up to levels of the anterior medullary velum and are present in all periventricular cell layers (Gonzalez and Munoz, 1988b; Roth et al., 1990). We find a n average of 110 Mes V cells in each tectal lobule in Rana ridibunda. Although the cell numbers in this nucleus vary enormously from animal to animal, comparable results were obtained for Xenopus (Lowe and Russell, 1984) and Rana pipiens (Kollros, 1984). In urodeles, a similar population was found (Gonzalez and Munoz, 198813). Both unipolar and multipolar cells with rounded or polygonal somata constitute the Mes V of Rana ridibunda. Actually, this could be the generalized composition for all vertebrates. Thus, although the morphology normally described for the Mes V cells is composed of a population of unipolar, large, oval-to-rounded neurons (mammals: Matsushita et al., 1981; Jacquin et al., 1983; Faccioli et al., 1985; Nozaki et al., 1985; birds: Wild and Zeigler, 1980; reptiles: Barbas-Henry and Lohman, 1986; Fernandez and Paz, 1984; amphibians: Roth et al., 1990; Gonzalez and M U ~ O Z1988b; , fish: New and Northcutt, 19841, multipolar cells have more recently been described in almost all animals studied (mammals: Gottlieb et al., 1984; Walberg, 1984; Carpra et al., 1985; Rokx et al., 1986; birds: Faccioli et al., 1985; reptiles: Szekely and Matsez, 1988; amphibians: Fritzsch and Sonntag, 1987; Lowe and Russell, 1984; fish: Witkovsky and Roberts, 1975; MacDonnell, 1980). The arrangement of Mes V cells found in the present study basically corresponds to a pattern of scattered cells with only some small cell clusters formed at the rostral pole of the tectum. No clear groups of cells with soma-soma appositions were observed. This seems to be a peculiarity of amphibians and strikingly contrasts with the close arrangement of Mes V cells present in other vertebrates (mammals: Hinrichsen and Larramendi, 1970; Lucchi et al., 1972; Alley, 1974; fish: Witkovsky and Roberts, 1976). In our study we attempted to determine a somatotopy in the distribution of Mes V cells. However our results failed to reveal differences in Mes V cell morphology and distribution depending on the treated nerve. The only somatotopy we found was related to the TRIGEMINAL MESENCEPHALIC NUCLEUS OF T H E FROG 175 Fig. 10. Diagram summarizing the arrangement of the central, descending, and peripheral branches of the Mes V fibers as found in the present study. Scale bar represents 1 mm. few cells in the caudal portion of the Mes V that were labeled via V3. The majority of Mes V cells labeled via V1, V2, or V3 are intermingled in the tectum. This result is in agreement with data in Xenopus with separate labeling of V3 and V1 (Lowe and Russell, 1984). However, in that case topographical relationship between soma position and axonal trajectory of Mes V cells was found. This correlation could not be elucidated in our experiments. The number of Mes V cells labeled from separate trigeminal branches reflects a higher population for V2, almost double that of V1 or V3. In Xenopus (Lowe and Russell, 1984), the number of Mes V cells labeled via V1 is similar to that of Rana ridibunda but the population obtained via the mandibular nerve was much higher in their experiments. However, no separate maxillary branch of the trigeminal nerve exists in Xenopus (Paterson 1939) and the number of Mes V cells via the large mandibular branch (67 approximately, Lowe and Russell, 1984) is probably similar to the population obtained after labeling V2 and V3 in Rana ridibunda. In ranid frogs, V2 innervates muscles in the jaw but also the musculus levator bulbi (Grusser and Grusser-Cornehls, 1976), thus the large population of Mes V cells with peripheral branches in V2 could represent those carrying proprioceptive impulses from the jaw and this extraocular muscle. Innervation of extraocular muscles by Mes V fibers in amphibians has been previously reported (Hiscock and Straznicky, 1982; Ciani et al., 1986) and was observed earlier in mammals (Alvarado-Mallart et al., 1975). The precise arrangement of the peripheral, central, and descending branches of the Mes V cells (terminology after Dacey, 1982, for reptiles) has been observed. Interestingly, the caudal extent of the descending branches only reaches two zones in the rostral rhombencephalon, i.e., the area of the trigeminal motor nucleus and another area just dorsal and lateral to it. Similar sites of termination have been described in the rat (Rokx et al., 1986) and the mallard (Arends and Dubbeldam, 1982). Within the area of the motor nucleus, terminals from the Mes V are present both among the cell bodies of the motoneurons and in an area just lateral and ventral to them. Investigations of the morphology of trigeminal motoneurons of anurans (Matesz and Szekely, 1978; Oka et al., 1987) have showed an extensive dendritic arborization ventrolaterally. This would suggest that in Rana ridibunda, Mes V terminals could be synapsing on both cell bodies and dendrites, as it has been also seen in several mammalian species (Hamos and King, 1980; Matesz, 1981; Luschei, 1987). In contrast, Rubinson (1970) only found axonal debris in the area of the lateral dendrites of trigeminal motoneurons after lesions in the tectum in Rana pipiens. The second field with Mes V terminals was located just dorsal to the rostral pole of the trigeminal motor nucleus. This corresponds to an area mediodorsal to the principal sensory nucleus of the trigeminus. In Rana esculenta (Matesz and Szekely, 1978), a similar location receives terminals after cobalt filling of the trigeminal nerve. However, their nature could not be 176 M. MUNOZ ET AL. established since labeled exteroceptive primary afferents and possible descending Mes V branches are close together. Similar difficulty was observed in our material in the experiment with HRP applied to the trigeminal nerve, and clear Mes V terminals were only recognized after tectal injections. This area with terminals from the Mes V in anurans, on the basis of cytological characteristics and topography, seems to be comparable to the supratrigeminal nucleus of mammals (Astrom, 1953; Torvik, 1956; Rokx et al., 1986) and birds (Arends and Dubbeldam, 19821, i.e., a n area of the brainstem with terminals of Mes V neurons axons arising as collaterals of a long descending pathway, the Probst trct. In the literature, several other termination sites for the Mes V descending branches have been proposed for anurans. The caudal rhombencephalon and spinal cord were considered to receive Mes V cell axon terminals (Rubinson, 1970; Matesz and Szekely, 1978; D’Ascanio et al., 1979; Grover and Griisser-Cornehls, 1976). However, HRP, cobalt, or fluorescent tracers injected into these structures failed to label Mes V cell bodies in the present study or in detailed studies of descending pathways to the spinal cord in Xenopus laevis and Rana esculenta (Ten Donkelaar et al., 1981; Toth et al., 1985). In addition, in Rana esculenta the trigeminal sensory nuclei were described to receive Mes V axons from collaterals of a long descending tract (Matesz and Szekely, 1978). In Rana ridibunda, we never observed such projections since when HRP was injected into those areas no Mes V cell bodies were labeled retrogradely. Injecting HRP into extraocular muscles, an additional projection of the Mes V neurons to the oculomotor nucleus has been described for Xenopus (Hiscock and Straznicky, 1982). However, in their experiments, the cell bodies with their dendritic arborization were retrogradely labeled in the oculomotor nucleus and probably, the analysis of terminals in that area led to a misinterpretation of the results. Projections to the oculomotor nucleus from the Mes V cells were not observed in our material and this concurs with previous work in amphibians (Rubinson, 1970; Matesz and Szekely, 1978; Gonzalez and Munoz 1988b). ACKNOWLEDGMENTS The authors wish to thank Drs. G.E. Meredith and W.J.A.J. Smeets for their continuous help and for improving the manuscript with comments and suggestions. We also thank Dr. E. Rausel for providing some of the photomicrographs and drawings. LITERATURE CITED Adams, J.C. 1981 Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. 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