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Distribution morphology and central projections of mesencephalic trigeminal neurons in the frog Rana ridibunda.

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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.
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