THE JOURNAL OF COMPARATIVE NEUROLOGY 497:542–565 (2006)
Afferent and Efferent Connections of
the Cerebellum of a Salmonid, the
Rainbow Trout (Oncorhynchus mykiss):
A Tract-Tracing Study
MÓNICA FOLGUEIRA,1 RAMÓN ANADÓN,2 AND JULIÁN YÁÑEZ1*
Department of Cell and Molecular Biology, Faculty of Sciences, University of A Coruña,
15071 A Coruña, Spain
2
Department of Ecology and Cell Biology, University of Santiago de Compostela, 15782
Santiago de Compostela, Spain
1
ABSTRACT
The connections of the cerebellum of the rainbow trout were studied by experimental
methods. The pretectal paracommissural nucleus has reciprocal connections with the cerebellum. Three additional pretectal nuclei project to both the corpus and valvula cerebelli, and
seem to receive cerebellar afferents. A large number of cells of the lateral nucleus of the
valvula project to wide regions of the cerebellum, including the valvula, the corpus, the
granular eminences, and the caudal lobe, whereas the contralateral inferior olive and scattered reticular cells project only to the corpus and valvula cerebelli. Afferents to the corpus
were also observed from the ventral tegmental nucleus, the “paraisthmic nucleus,” the
perilemniscal nucleus, the central gray, and the octavolateral area. Valvular afferents were
also observed from the torus semicircularis and the midbrain tegmental areas. In most cases
of cerebellar application, labeled fibers were seen in the thalamus, the pretectum, the torus
longitudinalis and torus semicircularis, the nucleus of the medial longitudinal fascicle, and
midbrain and rhombencephalic reticular areas. From the corpus cerebelli some fibers also
project to the posterior tubercle and the hypothalamus. Moreover, the granular eminences
project to the cerebellar crest. DiI application to most of the areas showing labeled fibers after
cerebellar tracer application led to the labeling of characteristic eurydendroid cells, mainly in
the valvula cerebelli and the caudal lobe. A few putative eurydendroid cells were labeled from
the octavolateralis regions. These results in a teleost with a generalized brain indicate
several differences with respect to the cerebellar connections reported in other teleost fishes
that have specialized brains. J. Comp. Neurol. 497:542–565, 2006. © 2006 Wiley-Liss, Inc.
Indexing terms: cerebellar connections; pretectal nuclei; octavolateral system; eurydendroid
cells; projections; teleosts
The cerebellum is a suprasegmental dorsal center located in the rostral rhombencephalon that plays a major
role in motor control and is involved in cognitive and
emotional functions (see Roberts et al., 2002; Rodrı́guez et
al., 2005). The cerebellum of teleost fishes consists of three
main regions: 1) the valvula cerebelli; 2) the corpus cerebelli, sometimes lobed; and 3) the caudoventral cerebellar region or vestibulolateral lobe (Larsell, 1967; Nieuwenhuys, 1967; Finger, 1983; Meek, 1992a). The corpus
cerebelli is the principal region of the cerebellum and is
visible as an unpaired dorsal protrusion of the brain. A
rostral extension of the corpus cerebelli into the midbrain
ventricle forms the valvula cerebelli, which is present only
in actinopterygian fishes (Kappers et al., 1936; Larsell,
1967; Nieuwenhuys, 1967) and is located in the midbrain
© 2006 WILEY-LISS, INC.
ventricle under the optic tectum. The caudoventral cerebellar region is frequently referred to as the vestibulolateral lobe because of its connections with the octavolateral
Grant sponsor: Spanish Science and Education Ministry; Grant number:
BFU2004-03144/BFI; Grant sponsor: Diputación Provincial de A Coruña
(to M.F.).
*Correspondence to: Julián Yáñez, Department of Cell and Molecular
Biology, Faculty of Sciences, University of A Coruña, 15071 A Coruña,
Spain. E-mail: juliany@udc.es
Received 28 July 2005; Revised 19 October 2005; Accepted 7 February
2006
DOI 10.1002/cne.20979
Published online in Wiley InterScience (www.interscience.wiley.com).
The Journal of Comparative Neurology. DOI 10.1002/cne
CEREBELLAR CONNECTIONS IN TROUT
543
region; it consists of the caudal lobe and paired granular
eminences (Pouwels, 1978a; Bass, 1982; Finger, 1983;
Nieuwenhuys and Pouwels, 1983; Bernochi et al., 1987;
Murakami and Morita, 1987).
The cellular organization of the corpus cerebelli of teleosts is known from a few Golgi (Cajal, 1911; Franz, 1911;
Nieuwenhuys, 1967; Nieuwenhuys and Nicholson,
1969a,b; Pouwels, 1978a– c; Murakami and Morita, 1987)
and electron microscopic studies (Kaiserman-Abramof
and Palay, 1969; Pouwels, 1978b,c). It appears similar to
that observed in other vertebrates and shows 1) an outer
molecular layer containing scattered stellate cells, dendrites of Purkinje cells and other large cerebellar neurons,
and parallel fibers originating from granule cells (no basket cells have been described in teleosts); 2) a layer of
large cells, the Purkinje cell or ganglionic layer, which is a
single sheet in salmonids; and 3) a granule cell layer,
containing both granular and Golgi cells (Pouwels,
1978b,c). The Purkinje cells of ray-finned fishes appear to
have connections only within the cerebellum. Teleosts
have no cerebellar nuclei, but show a group of specialized
cells, the eurydendroid cells that are intermingled with
Purkinje cells and send their axons out of the cerebellum
carrying cerebellar outputs (Finger, 1978b; Pouwels,
1978c; Murakami and Morita, 1987; Meek and Nieuwenhuys, 1991; Torres et al., 1992; Ikenaga et al., 2005).
Numerous granular cells occupy the lateral parts of the
corpus cerebelli, forming two masses called granular eminences that are visible on the outer surface of the brain.
Granular cells give rise to parallel fibers that run in the
cerebellar molecular layer and the cerebellar crest overlying the rhombencephalic lateral line region (Maler, 1974;
Bass, 1982; Meek, 1992a). Although the molecular, Purkinje cell, and granular cell layers are also present in the
valvula cerebelli and the caudal lobe, the order and arrangement of these three layers is apparently inverted
with respect to that described in the corpus cerebelli, as a
result of the invagination of the embryonic cerebellar
plate into the midbrain ventricle (see Pouwels, 1978a;
Meek and Nieuwenhuys, 1998, for details).
A number of studies of the connections of the teleost
cerebellum have been carried out in electroreceptive species, where the cerebellum is highly hypertrophied and
modified (Nieuwenhuys and Nicholson, 1969a; Bell, 1981;
Finger et al., 1978a,b, 1981; Szabo, 1983; Meek et al.,
1986a,b; Wullimann and Northcutt, 1990; Wullimann and
Rooney, 1990; Striedter, 1990). Electroreception is present
in ancient actinopterygian groups (cladistians and chondrosteans), was lost in neopterygians (Bullock et al.,
1983), and reappeared as a sensory modality during evolution in several groups of teleosts, such as the silurids,
mormyrids, and gymnotids (Wullimann, 1998). Gymnotids, mormyrids, and a small group of synodontid catfishes (Hagedorn et al., 1990) are actively electroreceptive
and detect changes in weak electrical fields generated by
their electric organs. Such fishes exhibit various specializations in the brain for the detection and processing of
electrosensory information, which are notably reflected in
the hypertrophy and complex anatomical and cellular organization of the cerebellum (Meek et al., 1986a,b; Meek
and Nieuwenhuys, 1998). The cerebellar organization of
nonelectroreceptive species probably represents the primitive condition of teleosts (Wullimann and Northcutt,
1989). Knowledge about the cerebellar connections in non-
Abbreviations
ATh
ATN
BC
CB
CC
CG
CPn
D
Dc
fr
GE
GR
H
HL
Hy
IN
IO
IP
IRN
LC
ll
lln
LR
LRN
LTN
LV
MB
ML
MLF
MN
MR
NI
nMLF
anterior thalamic nucleus of Holmgren (⫽ nucleus glomerulosus)
anterior tuberal nucleus
corpus cerebelli
cerebellum
cerebellar crest
rhombencephalic central gray
central pretectal nucleus
diffuse nucleus of the inferior hypothalamic lobe
central area of the dorsal telencephalon
fasciculus retroflexus
granular eminence
cerebellar granular layer
habenula
inferior hypothalamic lobe
hypothalamus
intermediate pretectal nucleus
inferior olive
interpeduncular nucleus
inferior reticular nucleus
locus coeruleus
lateral lemniscus
lateral line nerves
lateral recess
lateral reticular nucleus
lateral tuberal nucleus
lateral nucleus of the valvula
mammillary body
cerebellar molecular layer
medial longitudinal fascicle
medial octavolateralis nucleus
ventromedial nucleus of the midbrain reticular formation
nucleus isthmi
nucleus of the medial longitudinal fascicle
OB
OC
OLa
OT
PC
PG
pisth
PL
pll
PM
PP
PR
PT
PSp
PTN
R
Retic
RF
SG
SGN
SR
T
Td
Tegm
Th
TL
Tlo
TS
Tv
vAO
VC
VIIIn
VS
VT
olfactory bulb
optic chiasm
octavolateral region
optic tectum
paracommissural nucleus
preglomerular complex
paraisthmic nucleus
posterior hypothalamic lobe
perilemniscal nucleus
magnocellular preoptic nucleus
parvocellular preoptic nucleus
posterior recess
posterior tubercle
superficial pretectal nucleus, parvocellular part
posterior tuberal nucleus
superior raphe nucleus
reticular region
reticular formation
nucleus subglomerulosus
secondary gustatory visceral nucleus
superior reticular nucleus
telencephalon
dorsal thalamus
tegmentum
thalamus
torus lateralis
torus longitudinalis
torus semicircularis
ventral thalamus
ventral accessory optic nucleus
valvula cerebelli
octaval nerve
vagal viscerosensory column
ventral tegmental nucleus
The Journal of Comparative Neurology. DOI 10.1002/cne
544
M. FOLGUEIRA ET AL.
electroreceptive teleosts comes from experiments in cyprinids (Ito et al., 1982a; Wullimann and Northcutt, 1988,
1989; Ito and Yoshimoto, 1990; Wullimann and Meyer,
1993; Vonderschen et al., 2001; Ikenaga et al., 2002), a
“primitive” teleost (the osteoglossomorph Pantodon: Wullimann and Meyer, 1993; Wullimann and Roth; 1994), and
a few species of the most advanced groups of teleosts
(percomorphs: Ito et al., 1986; Murakami and Morita,
1987; Wullimann and Northcutt, 1988, 1989; Imura et al.,
2003; Xue et al., 2004). Most of these studies have centered on specific areas of the cerebellum.
The salmonids (Protacanthopterygii; trout and salmon)
are nonelectroreceptive teleosts with a generalized cerebellum. They occupy a key position in the teleost phylogeny, between primitive and advanced groups. Moreover,
the available studies in trout have proved very important
for comprehension of the cellular organization and morphogenesis of the cerebellum in teleosts (Pouwels,
1978a– c; Porteros et al., 1998). However, cerebellar connections in salmonids have not been investigated experimentally, with the exception of preliminary tract-tracing
data in the context of the organization of cholinergic systems (Pérez et al., 2000). The aim of the present study was
to perform a comprehensive analysis of the connections of
the cerebellum in a salmonid fish, the rainbow trout, using
a lipophylic tracer (DiI) that diffuses along cell membranes in fixed brain. This study continues a series of
studies on connections of the trout brain performed in our
laboratories (Folgueira et al., 2002, 2003, 2004a,b).
MATERIALS AND METHODS
We used 51 young adult rainbow trout (4 –7 cm in standard body length) obtained from a local fish farm (Piscifactoria Berxa, Mesı́a, Spain). The animals were deeply
anesthetized with 0.1% tricaine methane sulfonate (MS222; Sigma, St. Louis, MO) and transcardially perfused
with cold 4% paraformaldehyde in 0.1 M phosphate buffer
(PB) at pH 7.4. Brains were then carefully removed from
the skull and stored at 4°C in the same fixative until use.
All experiments conformed to the European Community
Guidelines on Animal Care and Experimentation.
The lipophylic tracer 1,1⬘-dioctadecyl 3,3,3⬘,3⬘-tetramethylindocarbocyanine perchlorate (DiI; Molecular
Probes, Eugene, OR) was applied using two different procedures. A small crystal of DiI was placed on the tip of an
electrolytically sharpened insect pin and directly inserted
into the brain under a stereomicroscope. The brain structures accessed by this procedure were superficial nuclei
and externally accessible brain areas, such as the valvula
(6 cases) and corpus cerebelli (8 cases), the caudal lobe (3
cases), the granular eminences (2 cases), the torus longitudinalis (2 cases), the cerebellar crests (5 cases), and the
octaval nerve (2 cases). In order to gain access in the cases
of valvular labeling, the caudal part of the optic tectum
was removed.
For DiI application to deeply located or less accessible
areas in reciprocal experiments, brains were sectioned on
a vibratome (Campden Instruments, Sileby, UK) after
being embedded in a block of 3% agarose. Some sections
were stained during the procedure with 0.1% methyl blue,
coverslipped with PB, and immediately observed under a
light microscope in order to determine the appropriate
level for the injection. After reaching the appropriate
level, the tracer was applied as above. Using this proce-
dure, DiI was applied to the following areas: central pretectal nucleus (2 cases), paracommissural nucleus (4
cases), intermediate pretectal nucleus (2 cases), ventral
accessory optic nucleus (2 cases), ventral tegmental nucleus (2 cases), nucleus of the medial longitudinal fascicle
(4 cases), torus semicircularis (2 cases), lateral nucleus of
the valvula (3 cases), and rostral spinal cord (2 cases).
In both procedures the tracer application area was
sealed with melted agarose and brains were left in the
dark for 2– 8 weeks in frequently renewed fresh fixative at
37°C. To assess the extension of the injection area, brains
were observed 24 hours after tracer application (Fig. 1).
After the transport period, transverse sections (50 ␮m
thick) were cut on a vibratome and mounted on gelatincoated slides. Sections were examined in an epifluorescence photomicroscope (Nikon E-1000) equipped with a
rhodamine filter set and photographed on Tmax-400 B&W
film (Kodak, Las Rozas, Spain). Negative films were digitally scanned (Epson, Tokyo, Japan), and the images were
inverted and balanced for brightness and contrast with
Adobe Photoshop (San Jose, CA) and then printed as positive.
Unless otherwise stated, the nomenclature for the different brain nuclei and areas was adopted from Northcutt
and Bradford (1980), Northcutt and Davis (1983), and
Wullimann and Northcutt (1988).
RESULTS
Connections of the valvula cerebelli
Valvular afferents. After DiI application to the rostrolateral region of the valvula cerebelli, retrogradely labeled cells were observed in the pretectum, isthmus, and
rhombencephalon, mainly on the ipsilateral side (Fig. 2).
At pretectal levels, some retrogradely labeled cells were
observed medial to the parvocellular superficial pretectal
nucleus (PSp) and intermingled with the optic fibers ascending to the tectum (Figs. 2A, 3A). These are the rostralmost labeled cells, belonging to the central pretectal
nucleus. In the dorsolateral periventricular pretectum, a
few labeled neurons were detected in the paracommissural nucleus (Figs. 2B–D, 3B). A few retrogradely labeled
cells were also observed in an intermediate pretectal nucleus (intermediate nucleus of Brickner, 1929; accessory
pretectal nucleus of Butler et al., 1991) (Figs. 2B–D, 3B),
which is located between the anterior thalamic nucleus of
Holmgren (posterior pretectal nucleus of Butler et al.,
1991) and the magnocellular superficial pretectal nucleus
(Garcı́a and Anadón, 1977), and in a region ventral to the
anterior thalamic nucleus (ventral accessory optic nucleus) (Fig. 2C). These pretectal cells were mostly pearshaped, although a few slightly polygonal perikarya were
also observed. The number of labeled cells was noticeably
smaller than that observed after DiI application to the
corpus cerebelli (see below). Occasional cells were labeled
in the torus semicircularis and in the rostral tegmentum
ventral to it (Figs. 2E, 3C,D). At caudal mesencephalic
levels, numerous small, round cells were strongly labeled
in the medial portion of the lateral nucleus of the valvula
(LV) (Figs. 2F,G, 3E).
In the corpus cerebelli, scattered granule-like cells were
labeled after DiI application to the valvula (Fig. 2I,J).
Furthermore, a small group of medium-sized labeled neurons was observed close to the granular eminences and
The Journal of Comparative Neurology. DOI 10.1002/cne
CEREBELLAR CONNECTIONS IN TROUT
545
Labeled fibers. After DiI application to the valvula cerebelli, labeled fibers were observed bilaterally, although the
ipsilateral component was predominant. Most labeled fibers
course in a compact pretecto-cerebellar tract (often referred
to as the mesencephalo-cerebellar tract) traversing the lateral nucleus of the valvula, or run in the brachium conjunctivum to decussate dorsally to the interpeduncular nucleus,
and their fibers ascend to midbrain and diencephalic levels,
or descend to isthmo-rhombencephalic levels (Fig. 2E–G).
Fibers leaving the pretecto-cerebellar tract coursed toward
the cerebellopetal pretectal nuclei (central, paracommissural, and intermediate pretectal nuclei) ipsilaterally, and a
few fibers also reach the torus semicircularis (Fig. 2B–G).
Labeled tracts coursing between the cerebellum and cerebellopetal pretectal nuclei probably include both cerebellar afferents and efferents (see below, Reciprocal experiments).
Fibers from the brachium conjunctivum reach the torus longitudinalis through the posterior commissure, the ipsilateral
thalamus, and, bilaterally, the nucleus of the medial longitudinal fascicle and the “nucleus ruber” of Oka et al. (1986)
(Figs. 2E,F, 3H). This “nucleus ruber” was identified in our
trout material after DiI application to one side of the spinal
cord, resulting in the labeling of a compact ipsilateral group
of rounded medium-sized cells ventrally to the nucleus of the
medial longitudinal fascicle. Accordingly, we considered that
it does not represent a true nucleus ruber and termed it the
ventromedial nucleus of the midbrain reticular formation
(see Discussion). In the cerebellum, labeled fibers from the
valvula were observed in the granular layer of the caudal
lobe and in the granular eminences (Fig. 2H,I). At caudal
levels of the corpus, a compact tract of fibers labeled from the
valvula course at rostral rhombencephalic levels along the
outer surface of the secondary gustatory nucleus (Fig. 2H).
These labeled fibers could be followed caudally to the level of
the inferior olive (olivocerebellar or spinocerebellar tract?).
Labeled fibers reaching the isthmic and rhombencephalic
medial reticular areas were also observed (Figs. 2H–K, 3I).
Connections of the corpus cerebelli
Fig. 1. Photomicrographs of a whole brain (A) and a sectioned
brain at the level of the midbrain (B) showing the small extension of
the tracer diffusion (arrows) after 24 hours of application of the DiI
crystal. Scale bars ⫽ 1 mm. [Color figure can be viewed in the online
issue, which is available at www.interscience.wiley.com.]
medial to the nucleus isthmi (Figs. 2H, 3F). In view of its
location, this cell group can be considered a caudal extension of the LV.
Occasional reticular neurons and their processes were
labeled at different rostrocaudal rhombencephalic levels
and also occasionally in the medial part of the octavolateralis region (Fig. 2I,J). In the contralateral caudoventral
rhombencephalon, a few small round cells were labeled in
the inferior olive (Figs. 2K, 3G).
Afferents. DiI application in the dorsolateral region of
the corpus cerebelli at different rostrocaudal levels led to
the labeling of cells and fibers in the pretectum and in
several nuclei of the midbrain and hindbrain (Fig. 4).
Although most of these cerebellopetal nuclei were labeled
bilaterally, the ipsilateral component was much more
marked. A number of cells and their processes were labeled in four pretectal nuclei: the central pretectal nucleus, the paracommissural nucleus, the intermediate pretectal nucleus, and the aforementioned ventral accessory
optic nucleus (Figs. 4A–D, 5A–E). Most of the labeled
pretectal cells were pear-shaped, but fusiform cells were
also observed. In addition, a few perikarya were labeled in
the torus longitudinalis (Fig. 4B–D). At caudolateral midbrain levels, a rather large group of medium-sized cells
was labeled in the ventral tegmental nucleus (Figs. 4E,F,
5F–H, 6A,B), located in the lateral corner of the mesencephalic tegmentum, ventral to the torus semicircularis, and
extending up to isthmic levels. The labeled perikarya of
this nucleus were mostly pear-shaped, but fusiform or
multipolar perikarya were also observed. A number of
small granule-like cells were conspicuously labeled in the
lateral nucleus of the valvula, including its prominent
rostral part (Figs. 4F,G, 6C,D). Occasional medium-sized
cells were also labeled in the trochlear nucleus (Figs. 4G,
6E), likely through the motor root traversing the cerebel-
The Journal of Comparative Neurology. DOI 10.1002/cne
546
M. FOLGUEIRA ET AL.
Fig. 2. Schematic drawings of transverse sections of trout brain (A–K) showing labeled perikarya
(solid circles) and fibers (dashes and lines) after DiI application to the valvula cerebelli. The shaded area
in F represents the application site. The levels of the sections are indicated in the lateral view of the
brain. For abbreviations, see list. Scale bar ⫽ 1 mm.
lum after decussating dorsally (see Discussion). Moreover,
a few labeled cells were observed in the pretrigeminal
central gray and in the locus coeruleus (Fig. 4H). Labeled
cells were also observed in a population lateral to the
nucleus isthmi (Figs. 4G, 6F), here termed the paraisthmic nucleus because of its position (see Discussion). A
small number of perikarya were labeled in the perilemniscal nucleus, close to the lateral lemniscus and sometimes intermingled with it (Fig. 6F). Some scattered
isthmo-rhombencephalic reticular neurons were also observed (Fig. 4G,I–K). In the caudal rhombencephalon, occasional retrograde labeled neurons were located in the
mechanosensory medial octavolateralis nucleus (McCormick, 1983) and in other octavolateral regions (Figs. 4J,
7A,B). Those cells of the medial octavolateralis nucleus
are pear-shaped or fusiform and show dendritic processes
entering the cerebellar crest overlying the nucleus. Large
and medium-sized, pear-shaped perikarya and their pro-
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CEREBELLAR CONNECTIONS IN TROUT
547
Fig. 3. Transverse sections through the diencephalon (A–C), mesencephalon (D,H), isthmus (E), and caudal rhombencephalon (F,G,I),
showing labeled cells and fibers after DiI application to the valvula
cerebelli. The midline is at the right, with the exception of G. A:
Retrogradely labeled cells (arrowheads) in the central pretectal nucleus. B: Labeled fibers and cells in rostral regions of the paracommissural (arrowhead) and intermediate pretectal nuclei (arrows). C: A
few scattered labeled cells (arrowheads) located in the mesencephalic
tegmentum. Asterisk: the pretecto-cerebellar tract. D: A few labeled
cells in the torus semicircularis (arrowhead). Note the pretectocerebellar tract (asterisk) and anterogradely labeled fibers in the
mesencephalic tegmentum. E: Labeled granular cells (arrowheads)
and their processes (arrows) in the lateral nucleus of the valvula. F:
Retrogradely labeled cells (arrowhead) in a caudal part of the lateral
nucleus of the valvula. Note anterogradely labeled fibers (arrow) in
the caudal lobe and in the cerebellar peduncle (open arrow). G: Retrogradely labeled cell (arrowhead) in the contralateral inferior olive,
and anterogradely labeled fibers (arrow). H: Anterogradely labeled
fibers in the nucleus of the medial longitudinal fascicle (arrow). The
outlined arrow points to labeled fibers in the ventromedial nucleus of
the midbrain reticular formation. I: Anterogradely labeled fibers (arrow) in the caudal rhombencephalon. Note a lateral and a medial tract
(arrowheads). For abbreviations, see list. Scale bars ⫽ 100 ␮m in
A,C–E; 75 ␮m in B,G,H; 25 ␮m in F; 200 ␮m in I.
cesses were also labeled in the inferior reticular nucleus
(Figs. 4J, 7A), some probably corresponding to the nucleus
of the commissure of Wallenberg (1907). The contralateral
inferior olive showed many retrogradely labeled cells
(Figs. 4K, 7C).
Within the cerebellum, some granule-like cells were
labeled in the caudal part of the corpus cerebelli and, in
one experiment, labeled stellate cells were observed on the
contralateral side close to the DiI application area (Fig.
7E–G). The dendrites of stellate cells radiate in all directions from the cell body. In this experiment, some Purkinje
cells showing typical dendritic trees (Fig. 7D) together
with some granule cells could be observed slightly caudal
to the DiI application point.
Labeled fibers. Labeled fibers from the corpus cerebelli were observed predominantly contralateral to the
tracer application side. Two main tracts, the pretectocerebellar tract and the brachium conjunctivum, were la-
The Journal of Comparative Neurology. DOI 10.1002/cne
548
M. FOLGUEIRA ET AL.
Fig. 4. Schematic drawings of transverse sections (A–K) showing labeled perikarya (solid circles) and
fibers (dashes and lines) after DiI application to the corpus cerebelli. The shaded area in I represents the
typical application site. The levels of the sections are indicated in the lateral view of the brain. For
abbreviations, see list. Scale bar ⫽ 1 mm.
beled as described for the valvula (Fig. 4G,H). At rostral
levels, anterograde-labeled fibers coursing in the pretectocerebellar tract seem to innervate the four pretectal nuclei
which we denominate the corpopetal nuclei (the central,
paracommissural, and intermediate pretectal nuclei, and
the nucleus ventral to the anterior thalamic nucleus), as
well as the ventral tegmental nucleus (Fig. 4A–F). Nevertheless, the large number of perikarya and their processes
labeled in these nuclei make it difficult to see terminal
fields. Fibers labeled from the corpus and decussating in
the posterior commissure were observed entering the
torus longitudinalis (Figs. 4B, 5C). A few labeled fibers
were observed bilaterally in the torus semicircularis (Figs.
4E, 5H), the contralateral component showing a higher
density of labeled fibers. Labeled fibers coursing in the
brachium conjunctivum reach contralaterally the ventromedial nucleus of the midbrain reticular formation, the
nucleus of the medial longitudinal fascicle, the dorsal and
The Journal of Comparative Neurology. DOI 10.1002/cne
CEREBELLAR CONNECTIONS IN TROUT
Fig. 5. Transverse sections through the diencephalon (A–F) and
mesencephalon (G–H) of trout, showing structures labeled after
tracer application to the corpus cerebelli. The midline is at the right in
D,E, H, at the left in B,G. A: Retrogradely labeled cells (arrowheads)
bilaterally in the central pretectal nucleus, and a few anterogradely
labeled fibers (arrows) reaching the contralateral thalamus. B: Detail
of labeled cells in the ipsilateral central pretectal nucleus (arrowheads) after application of a minute DiI crystal to the corpus cerebelli.
C: A few retrogradely labeled cells in the contralateral pretectal area
(arrowheads) and in the torus longitudinalis (outlined arrowhead).
Anterogradely labeled fibers reaching the thalamus (outlined arrowheads) and the torus longitudinalis (arrows). Note the tracts entering
the torus longitudinalis. D: Labeled perikarya in the paracommissural nucleus, the intermediate pretectal nucleus (arrowheads), and
the ventral accessory optic nucleus (outlined arrowhead). E: Detail of
labeled perikarya in the paracommissural nucleus (arrowheads) and
the intermediate pretectal nucleus (arrows). F: Labeled fibers reach-
549
ing the rostral torus semicircularis (outlined arrow), the nucleus of
the medial longitudinal fascicle, the ventromedial nucleus of the midbrain reticular formation (arrow), and the posterior tubercle (arrowhead). Note the retrogradely labeled cells in the ventral tegmental
nucleus (outlined arrowhead). G: Contralateral to the DiI application
side, a few labeled cells in the ventral tegmental nucleus (arrowhead)
and anterogradely labeled fibers innervating the medial longitudinal
fascicle, the ventromedial nucleus of the midbrain reticular formation
(arrow), the posterior tubercle, and the inferior hypothalamic lobe
(outlined arrow). Note the compact pretecto-cerebellar tract located
ventral to the torus semicircularis. H: Labeled cells in the ipsilateral
ventral tegmental nucleus (arrowhead). Note a few labeled fibers and
cells in the mesencephalic tegmentum (outlined arrowhead), and fibers reaching the posterior tubercle (arrow). For abbreviations, see
list. Scale bars ⫽ 150 ␮m in A,B,D; 200 ␮m in C,G,H; 100 ␮m in E; 400
␮m in F.
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Fig. 6. Transverse sections through the mesencephalon (A–E) and
isthmus (F) showing structures labeled after DiI application to the
corpus cerebelli. The midline is at the right in A,E-F, at the middle in
B, and at the left in C. A: Detail of the labeled perikarya (arrowheads)
in the ventral tegmental nucleus. B: Retrogradely labeled cells in the
lateral nucleus of the valvula (arrowheads) and in the ventral tegmental nucleus (arrow). C: Detail of labeled cells (arrowheads) in the
lateral nucleus of the valvula and fibers (arrows) reaching the torus
semicircularis. D: Retrogradely labeled perikarya in the lateral nucleus of the valvula (arrow) and in the ventral tegmental nucleus
(arrowheads). E: Detail of two labeled perikarya in the region of the
trochlear nucleus (arrowheads). F: Retrogradely labeled cells (arrowheads) in the paraisthmic nucleus. Note a labeled cell (arrow) belonging to the perilemniscal nucleus, which is intermingled with the
lateral lemniscus. For abbreviations, see list. Scale bars ⫽ 100 ␮m in
A,F; 300 ␮m in B; 150 ␮m in C,D; 50 ␮m in E.
ventral thalamus, the tuberal nuclei, the posterior tubercle, and the medial part of the inferior hypothalamic lobe
(Figs. 4A–F, 5A,C,F–H). Fibers coursing to rhombencephalic levels could be followed to the superior and intermediate reticular formation (Fig. 4G–I). At the level of the
granular eminences, a compact tract of labeled fibers was
observed running toward ventrolateral rhombencephalic
regions (Fig. 4H). As mentioned for the valvula cerebelli,
some of the labeled fibers originate in the inferior olive.
In the dorsal molecular layer of the corpus cerebelli,
varicose parallel fibers run to the contralateral side close
to the DiI application area (Fig. 7D–G). Occasionally, the
typical T-shaped branching of parallel fibers could be observed.
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Fig. 7. Transverse sections through the medulla oblongata (A–C)
and corpus cerebelli (D–G) showing structures labeled after DiI application to the corpus cerebelli. Photomicrographs D–G show areas
close to the point of DiI application. The midline is at the left in B,C,
at the right in D–G. A: Reticular (arrowhead) and octavolateral (arrows) labeled cells in the caudal rhombencephalon. B: Detail of a
labeled octavolateral cell extending its dendrite into the cerebellar
crest. C: Retrogradely labeled cells (arrowhead) in the contralateral
inferior olive. D: Putative Purkinje cell (arrowhead) near the point of
tracer application showing its dendritic tree entering the molecular
layer (arrow), and labeled parallel fibers (outlined arrowhead). E:
Labeled stellate cells (arrowheads) and parallel fibers (arrows) in the
molecular layer of the contralateral side close to the point of DiI
application. F: Detail of a labeled stellate cell. G: Labeled parallel
fibers showing the typical T-shaped branching (arrows). For abbreviations, see list. Scale bars ⫽ 150 ␮m in A; 100 ␮m in B,C; 50 in D,F;
75 ␮m in E; 30 ␮m in G.
Connections of the caudal cerebellum
(caudal lobe and granular eminences)
observed in the paracommissural nucleus, but no labeled
cells were observed in other pretectal areas (Figs. 8A,B,
9A). A few cells were weakly labeled in medial regions of
the lateral nucleus of the valvula (Figs. 8D,E, 9B).
Close to the DiI application point a number of labeled
large and medium-sized cells were scattered in the medial
region of the caudal lobe (Fig. 8F,G). Some of these cells
were Purkinje cells, but the possibility that other cell
Caudal lobe. Afferents. DiI application to the caudal
lobe led to the labeling of both cells and fibers at diencephalic, mesencephalic, and cerebellar levels (Fig. 8). However, the number of labeled nuclei was smaller than that
observed after DiI application to the valvula and corpus
cerebelli. At diencephalic levels a few labeled cells were
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M. FOLGUEIRA ET AL.
Fig. 8. Schematic drawings of transverse sections (A–H) showing labeled perikarya (solid circles) and
fibers (dashes and lines) after DiI application to the caudal lobe. The shaded area in G represents the
application site. The levels of the sections are indicated in the lateral view of the brain. For abbreviations,
see list. Scale bar ⫽ 1 mm.
types were labeled (eurydendroid cells, Golgi cells) cannot
be ruled out. Occasional stellate cells were labeled in the
molecular layer of the caudal lobe.
Labeled fibers. As described for the valvula and corpus
cerebelli, labeled fibers were observed in the pretectocerebellar tract and decussating in the brachium conjunctivum dorsally to the interpeduncular nucleus (Fig. 8C–
E). Labeled fibers were observed in the paracommissural
nucleus, the nucleus of the medial longitudinal fascicle,
and the ventromedial nucleus of the midbrain reticular
formation (Fig. 8A–C). At rhombencephalic levels, labeled
varicose fibers were observed coursing in the cerebellar
crest overlying the octavolateral area (Fig. 8H). In the
caudal lobe close to the tracer application point, anterogradely labeled fibers coursed through the molecular
layer. Two small tracts of labeled fibers coursing ventral to
the descending root of the trigeminal nerve could be followed to the level of the obex (Figs. 8H, 9C). Neither the
origin nor the final destination of those fibers could be
determined.
Granular eminences. Afferent neurons. DiI application to the granular eminences led to the labeling of cells
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Fig. 9. Transverse sections through the diencephalon (A), mesencephalon (B), and rhombencephalon (C) after DiI application to the
caudal lobe. The midline is at the right. A: Anterogradely labeled
fibers and a few labeled cells (arrowhead) in the paracommissural
nucleus. B: Faintly labeled cells (arrowheads) and a few labeled fibers
in the lateral nucleus of the valvula. C: Two labeled tracts (arrowheads) at caudal rhombencephalic levels. Scale bars ⫽ 125 ␮m in A,B;
250 ␮m in C.
mostly in the lateral nucleus of the valvula and at
rhombencephalic levels (Fig. 10). Some cells were labeled
in the lateral nucleus of the valvula (Fig. 10A,B). A group
of medium-sized perikarya and their processes were bilaterally labeled ventral to the secondary gustatory nucleus,
probably corresponding to the superior reticular nucleus
(Figs. 10C,D, 11A,B). Labeled fibers of these cells cross the
ventral midline at intermediate dorsoventral levels (Figs.
10C,D, 11A). Occasional perikarya were bilaterally labeled in the medial octavolateralis nucleus and in the
locus coeruleus (Figs. 10C,E, 11C,D). Cells of the medial
octavolateralis nucleus give rise to arcuate fibers crossing
the midline rather dorsally (Figs. 10E, 11C). Some reticular cells were labeled in the lateral reticular region at the
level of the vagal viscerosensory lobe (Figs. 10E,F, 11E).
These bipolar or multipolar cells give rise to conspicuous
commissural axons crossing the midline at intermediate
dorsoventral levels. No cells were labeled in the inferior
olive in these experiments. In addition, primary fibers of
the anterior and posterior lateral line nerves were labeled
(Fig. 10E). These fibers were not traced to the cell
perikarya in the corresponding ganglia where they originate.
Labeled fibers. Fairly abundant labeled fibers were observed in the granular layer of the valvula and corpus
cerebelli (Figs. 10A–C, 11F) and, at caudal cerebellar levels, varicose parallel fibers reached the molecular layer of
the caudal lobe (Figs. 10D, 11H). Numerous labeled fibers
course in thick bundles that were observed crossing to the
contralateral side, forming a conspicuous cerebellar commissure in caudal regions of the corpus (Figs. 10D, 11G).
In the cerebellar crest overlying the medial nucleus of the
octavolateral area there were numerous thin varicose fibers coursing longitudinally (Figs. 10E, 11I), which here
were interpreted as cross-sections of parallel fibers arising
from granule cells of the granular eminences.
between the corpus and valvula (Fig. 12A) and gives off
fibers with small swellings coursing in the granular layer
of the valvula, corpus cerebelli, and caudal lobe (Fig. 12B).
The morphology of these fibers is similar to the mossy
fibers revealed in the trout cerebellum by Golgi methods
(Pouwels, 1978b). Some labeled fibers or collaterals of the
pretecto-cerebellar tract reached the lateral nucleus of the
valvula (Fig. 12C,D). In this nucleus, two types of terminal
were observed, thin fibers and cup-shaped or round fibers
closely associated with a single valvular neuron. This latter type of terminal was more faintly labeled than the thin
fibers.
DiI application to these nuclei also led to labeling of
scattered large perikarya identified as eurydendroid cells,
mostly distributed in the valvula cerebelli and caudal lobe,
but also in the corpus cerebelli. The analysis of other
connections of pretectal nuclei is outside the scope of this
study, and they will not be further described.
Ventral tegmental nucleus. After DiI application to
the ventral tegmental nucleus, anterogradely labeled fibers course caudalward in the pretecto-cerebellar tract to
the cerebellum and terminate as mossy fibers in the granular layer of the valvula, corpus cerebelli, and caudal lobe.
A number of eurydendroid cells were labeled in the valvula cerebelli. In addition, a number of labeled cells were
observed in the optic tectum.
Nucleus of the medial longitudinal fascicle. After
tracer application to the cerebellum, anterogradely labeled fibers were observed in the region of the nucleus of
the medial longitudinal fascicle and the ventromedial nucleus of the midbrain reticular formation. In order to characterize the cerebellar cells projecting to these nuclei, we
used DiI applications to the nuclei, with long incubation
periods (more than 2 months) to allow the tracer to fill the
large eurydendroid perikarya and their processes. Although DiI application to other brain areas also labeled
eurydendroid cells, this procedure more effectively revealed the morphology of these cells (Fig. 12E,F). Labeled
eurydendroid cells were located below the Purkinje cell
layer and distributed throughout all the regions of the
cerebellum, but mainly in the contralateral valvula cerebelli and caudal lobe. In the valvula and corpus cerebelli,
eurydendroid cells appeared either as fusiform or multipolar, all showing smooth slender dendrites, scarcely
branched, ascending toward the molecular layer. In the
Reciprocal experiments
Precerebellar pretectal nuclei. Experiments of DiI
application to the central pretectal nucleus, the paracommissural nucleus, the intermediate pretectal nucleus, or
the ventral accessory optic nucleus led to labeling of similar structures in the cerebellum. Labeled fibers were observed coursing in the pretecto-cerebellar tract. The compact pretecto-cerebellar tract enters the cerebellum
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Fig. 10. Schematic drawings of transverse sections (A–F) showing labeled perikarya (solid circles)
and fibers (dashes and lines) after DiI application to the granular eminence. The shaded area in D
represents the application site. The levels of the sections are indicated in the lateral view of the brain.
For abbreviations, see list. Scale bar ⫽ 1 mm.
ventral caudal lobe, a number of eurydendroid cells with
fusiform or round somata were labeled. Round cells give
rise to a single dendrite that branches in the molecular
layer rather distant from its origin. Some granule-like
cells were also labeled in the granular eminences.
Torus longitudinalis. DiI application to torus longitudinalis led to labeling of neurons in the optic tectum, the
eminentia thalami, and a few pretectal nuclei (paracommissural, intermediate) (not shown). In the cerebellum,
some labeled fibers were observed, but not labeled
perikarya.
Torus semicircularis. Application of DiI to the torus
semicircularis (approached from rostral) led to labeling of
a number of neurons in the octavolateralis region (crest
cells, medial octavolateralis nucleus, medial region of the
ventral octavolateralis nucleus), a few reticular cells,
some cells associated with the lateral lemniscus (perilemniscal nucleus), and a few large cells located in the granular layer of the corpus cerebelli.
Lateral nucleus of the valvula. DiI application to the
lateral nucleus of the valvula was done via a caudal ap-
proach in sectioned brain, allowing study of rostral connections only. These experiments led to the labeling of
cells in precerebellar structures at pretectal, isthmic, and
rhombencephalic levels. In the pretectum, most retrogradely labeled cells were observed in the central pretectal
nucleus, and occasionally in the paracommissural and
intermediate pretectal nuclei (Fig. 13A–C) and ventral
accessory optic nucleus. In the central and paracommissural nuclei, labeled cells are pear-shaped and exhibit
oriented dendritic trees. In addition, in the diencephalon
we observed some labeled cells in the preoptic nucleus,
occasional labeled cells in the dorsomedial thalamus (central posterior nucleus), a few scattered large labeled cells
in the diffuse nucleus of the inferior hypothalamic lobe
(Fig. 13D), and some medium-sized labeled cells in the
nucleus of the posterior tubercle. Numerous retrogradely
labeled cells were observed contralaterally in the LV (Fig.
13E). These small cells show a globular morphology, lacking complex dendritic trees. In the midbrain, occasional
labeled cells were observed in the laminar nucleus that
lies rostromedial to the torus semicircularis. In one tract-
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Fig. 11. Transverse sections through the rhombencephalon (A–
E,I) and cerebellum (F–H) after DiI application to the granular eminence. The midline is at the right, with the exception of G. A: Labeled
cells (arrowheads) of the superior reticular nucleus and labeled fibers
in the granular eminences (arrow) and in the rhombencephalic tegmentum crossing to the contralateral side (outlined arrow). B: Detail
of the reticular cell perikarya (arrowheads). C: Labeled cells (arrowheads) in the medial octavolateralis nucleus and labeled fibers crossing to the contralateral side (arrow). D: Labeled multipolar cell of the
locus coeruleus. E: Labeled lateral reticular cells (arrowheads) in the
caudal rhombencephalon and anterogradely labeled fibers. F: Anterogradely labeled fibers (arrows) in the ventrolateral region of the
valvula cerebelli. G: Labeled fiber bundles (arrowhead) crossing to the
contralateral side at caudal regions of the corpus. H: Varicose labeled
fibers (arrows) reaching the ventral part of the caudal lobe. I: Cross
section through the cerebellar crest showing thin varicose fibers (arrow). Note the labeled cell in the medial octavolateralis nucleus (arrowhead). For abbreviations, see list. Scale bars ⫽ 125 ␮m in B,C,H;
175 ␮m in A,F; 75 ␮m in D,E,I; 375 ␮m in G.
tracing experiment a number of ipsilateral eurydendroid
cells were labeled dorsally in the valvula cerebelli (Fig.
13F). At caudal rhombencephalic levels a few labeled cells
were observed in medial reticular regions (Fig. 13G).
Labeled fibers were observed in the lateral nucleus of
the valvula, on the contralateral side. Labeled fibers were
also observed reaching the walls of the posterior hypothalamic lobe (Fig. 13H), the inferior hypothalamic lobe, the
torus longitudinalis (Fig. 13I), and the dorsal and ventral
thalamus.
Cerebellar crest. DiI application to the cerebellar
crest overlying the octavolateral area led to the labeling of
a large number of granular cells in the granular eminences, mostly on the ipsilateral side (Fig. 14A). In some
experiments in which DiI affected the deeper regions of
the octavolateral region, large retrogradely labeled
perikarya were also observed in the caudal lobe (Fig. 14B).
Although the dendritic trees of these cells were generally
very lightly stained, in some instances these cells were
well-stained, showing dendritic trees in the molecular
layer (Fig. 14C). These cells were interpreted as eurydendroid cells. Tracer application to the cerebellar crest led to
the labeling of large pear-shaped crest cells with
perikarya located in the medial octavolateralis nucleus
(Fig. 14D). These neurons were labeled even in cases of
very superficial tracer application. Occasional labeled stellate cells were observed within the ipsilateral cerebellar
crest (Fig. 14E). Labeled cells were also observed in the
intermediate and inferior reticular formation (Fig. 14F).
In one experiment, retrogradely labeled cells were observed in the locus coeruleus and the superior reticular
nucleus (Fig. 14G).
Anterograde labeled arcuate fibers were observed reaching the contralateral medial octavolateralis nucleus and
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Fig. 12. Transverse sections through the corpus cerebelli
(A,B,E,F) and mesencephalon (C,D) showing labeled structures after
DiI application to the central pretectal nucleus (A), paracommissural
nucleus (B,C), and nucleus of the medial longitudinal fascicle (E,F). A:
Labeled fibers of the pretecto-cerebellar tract (arrow) in the caudal
corpus cerebelli. B: Labeled fibers (arrows) at the caudal corpus cerebelli. C: Retrogradely labeled cell (arrowhead) in the lateral nucleus
of the valvula (LV). Note labeled fibers (arrows) embracing cells of the
LV. D: Detail of a labeled fiber embracing a cell of the LV. E: Labeled
multipolar eurydendroid cell (arrowhead) in the ganglionic layer of
the valvula cerebelli. Note dendritic processes (arrows) entering the
molecular layer. F: Labeled eurydendroid cells in the caudal lobe
showing pear-shaped and bipolar (arrowheads) morphologies. The
arrow points to dendritic processes. For abbreviations, see list. Scale
bars ⫽ 150 ␮m in A; 100 ␮m in B; 75 ␮m in C; 30 ␮m in D,F; 40 ␮m
in E.
forming a band of terminals parallel to the crest (Fig.
14H); in addition, arcuate fibers or branches of these fibers
ascended in the contralateral lateral lemniscus to the
dorsolateral part of the torus semicircularis (mechanosensory region) (Fig. 14,I).
Octaval nerve. Application of DiI to the octaval nerve
led to labeling of fibers coursing caudally and rostrally in
the lateral part of the octavolateralis region. A bundle of
labeled fibers coursed toward the cerebellum passing over
the secondary gustatory nucleus. Fibers of this tract were
followed to the granular layer of the corpus cerebelli and
caudal lobe.
labeling far from the area of interest. The crystals used by
us are very small, typically 20 –50 ␮m in diameter, as
shown by conspicuous small red points seen after a short
time of incubation and the little scar seen in the first few
sections. In these experiments the extension of the labeled
region after 24 hours of application (see Fig. 1) is very
small and limited to the area of interest. Despite the
number of experiments done, in large centers such as the
cerebellum some projections limited to a small cerebellar
region might have been overlooked.
Second, DiI diffuses both retrogradely and anterogradely from the branching points of fibers (tracts, collateral fibers), which can lead to wrong conclusions regarding
the origin of labeled fibers (this does not apply to labeled
perikarya). This problem affects primarily to the putative
cerebellar efferents revealed after DiI application to the
cerebellum. It can be solved by reciprocal experiments of
application of DiI to the presumed targets: only confirmed
are those that reveal labeled perikarya in the cerebellum.
Since it was not possible to make applications to all possible targets, when experiments were done we distinguished between confirmed and nonconfirmed projections.
Third, tracts passing through a nucleus or region are
labeled after application of DiI. The problem surfaces
when trying to distinguish target nuclei from regions of
passage of fibers. To a different extent, similar problems
may affect the results of experimental studies based on
the application of other types of neuronal tracer (HRP,
biocytin, dextran amine). In charts, only terminal fields
clearly recognizable by the presence of terminal arbors or
DISCUSSION
Methodological considerations
The DiI methodology used in fixed tissue is highly sensitive (Godement et al., 1987; Holmqvist et al., 1992) and
offers the possibility of tracing the connections of very
small structures, even if they are of very difficult access
(Yáñez et al., 1996, 1997; Folgueira et al., 2002, 2003,
2004a,b). However, results of DiI-tracing experiments in
the central nervous system should always be interpreted
with caution (see Folgueira et al., 2004a). Here, we discuss
some problems that may affect the interpretation of results of cerebellar connections.
First, because DiI is applied as a water-insoluble crystal, diffusion is primarily limited to the area immediately
adjacent to the crystal, the amount of tracer incorporated
into cell membranes in this area is enough to show intense
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Fig. 13. Transverse sections through the diencephalon (A–
C,F,H,I), mesencephalon (D), valvula cerebelli (E) and rhombencephalon (G) showing labeled structures after DiI application to the lateral
nucleus of the valvula. The midline is at the right with the exception
of E. A: Labeled cells in the ipsilateral central pretectal nucleus
(arrowheads) and in the paracommissural nucleus (arrow). B: Retrogradely labeled cells (arrowheads) in the central pretectal nucleus at
a level rostral to A. C: Detail of a labeled central pretectal neuron
(arrowhead) and its dendritic processes (arrow). D: Labeled cells
(arrowheads) in the diffuse nucleus of the inferior hypothalamic lobe.
E: Labeled granular cells (arrowheads) in the contralateral lateral
nucleus of the valvula and labeled fibers (arrow). F: Labeled eurydendroid cells (arrowheads) in the ipsilateral valvula cerebelli. G: A
labeled reticular cell (arrowhead) and a compact tract of labeled fibers
(asterisk) at caudal rhombencephalic levels. H: Anterogradely labeled
fibers (arrows) reaching the walls of the posterior hypothalamic lobe
(PL). I: A retrogradely labeled cell (arrowhead) in the pretectal area
and labeled fibers (arrows) in the torus longitudinalis. For abbreviations, see list. Scale bars ⫽ 125 ␮m in A,D,F,H; 175 ␮m in B,E,G; 35
␮m in C; 200 ␮m in I.
beaded fibers were represented, but this does not rule out
that other centers can be contacted by en passant labeled
fibers. In teleosts, myelinated fibers may have regions
with short myelin segments forming small en passant
contacts with postsynaptic structures just at the nodes
without any apparent branching (Waxman, 1970, 1971).
Fourth, in trout DiI generally fails to diffuse efficiently
along highly myelinated pathways, which may lead to
false-negative results (Folgueira et al., 2004a). To minimize this problem, we used small immature specimens
(4 –7 cm in length) that show poorly myelinated pathways.
In terms of brain morphology and connections, these specimens should be considered small adults, not developing
stages. Brain morphogenesis is completed in trout of about
2.5 cm in length (Candal et al., 2005a,b), and experimental
studies of the retinotectal projection indicate that the
branching pattern is completed in trout of about 2.6 cm in
length (Mansour-Robaey and Pinganaud, 1996). Moreover, available studies of axonal development in the retinotectal and somatomotor systems of zebrafish have indicated that they do not form an excess of fiber connections
to be further trimmed and remodeled (Myers et al., 1986;
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Fig. 14. Transverse sections through the cerebellum (A–C),
rhombencephalon (D–H) and mesencephalon (I) showing labeled
structures after DiI application to the cerebellar crest. The midline is
at the left in H. A: Labeled granular cells (arrowheads) in the granular eminences. B: Large labeled perikarya in the caudal lobe (arrowheads). C: Labeled eurydendroid cell (arrowhead) and granule cells
(arrow) in the valvula cerebelli. D: Labeled crest cells (arrowhead) in
the ipsilateral medial octavolateralis nucleus close to the DiI application point. Note their dendritic processes entering the cerebellar
crest (arrow). E: Labeled stellate cells (arrowheads) in the cerebellar
crest close to the DiI application point. F: Labeled reticular cells in the
inferior reticular nucleus (arrowheads). G: Labeled reticular cells
(arrowheads) in the superior reticular nucleus. H: Anterogradely labeled fibers coursing to the contralateral medial octavolateralis nucleus (arrowhead) and octaval areas (arrow). I: Anterogradely labeled
fibers in the mechanosensory area of the torus semicircularis (arrow).
For abbreviations, see list. Scale bars ⫽ 150 ␮m in A,F,I; 100 ␮m in
B,D,E,H; 300 ␮m in C,G.
Liu and Westerfield, 1990; Stuermer et al., 1990). However, developmental changes of connections have been
documented in visual systems of some teleosts (Ebbesson,
1980; Ebbesson et al., 1988; Fritzsch and Wilm, 1992;
Rodrı́guez et al., 2003). Although it is expected that the
cerebellar connections revealed in 4 –7 cm long trout were
representative of those occurring in adults, it is possible
that some late-appearing cerebellar connections were not
demonstrated, or that some of the connections observed in
these trout juveniles were pruned in the later stages.
Comparison of the present results with those obtained in
the cerebellum of other teleosts with other tracing methods indicates that the present approach reveals most the
cerebellar connections reported in teleosts (see below).
generally in groups showing notable hypertrophy of sensory systems, such as the cyprinids, catfishes, and electric
fishes. In these fishes the cerebellar system has evolved
specialized nuclei and differentiations in order to process
specialized sensory information. This study considers cerebellar connections in the rainbow trout, a salmonid with
a generalized brain lacking hypertrophied sensory systems. The hodology of the salmonid cerebellum revealed
here may be representative of generalist teleosts. The
connections of the different regions of the trout cerebellum
are schematically represented in the Figure 15.
Connections of the corpus cerebelli. The results obtained after DiI application to the corpus cerebelli of the
rainbow trout confirm and extend previous data by Pérez
et al. (2000). After DiI application to the corpus cerebelli,
retrogradely labeled cells were observed in various brain
regions, mainly ipsilaterally. Most cells afferent to the
corpus and valvula cerebelli were located in the pretec-
Cerebellar connections
Most previous studies of cerebellar connections in teleosts have focused on specific areas of the cerebellum,
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559
tum, mesencephalic tegmentum, and rhombencephalon,
as described in other species of nonelectroreceptive (Wullimann and Northcutt, 1988, 1989; Xue et al., 2004) and
electroreceptive teleosts (Meek et al., 1986a,b; Striedter,
1990). These results recall the connections observed in a
primitive bony fish (Huesa et al., 2003).
At pretectal levels, retrogradely labeled cells were observed in the central pretectal nucleus, the paracommissural nucleus, Brickner’s intermediate pretectal nucleus
(⫽ accessory pretectal nucleus of Butler et al., 1991) and
the (ventral) accessory optic nucleus. Cerebellopetal neurons have been observed in the pretectum of several teleosts, including electroreceptive species (Finger, 1978a;
Grover and Sharma, 1981; Ito et al., 1982a; Meek et al.,
1986b; Murakami and Morita, 1987; Wullimann and
Northcutt, 1988, 1989; Ito and Yoshimoto, 1990; Wullimann and Meyer, 1993; Ikenaga et al., 2002; Imura et al.,
2003; Xue et al., 2004), and also in nonteleostean fishes
(elasmobranchs: Fiebig, 1988; chondrosteans: Huesa et
al., 2003). The intermediate (accessory) pretectal nucleus
of salmonids receives retinal projections (Shiga et al.,
1987; unpubl. results) and projects to the cerebellum
(present results), like the dorsal accessory optic nucleus of
other teleosts (Wullimann and Northcutt, 1988, 1989),
and probably corresponds to the dorsal accessory optic
nucleus of other species that projects to the cerebellum.
Cerebellar afferents from the central pretectal nucleus,
the ventral accessory optic nucleus, and the paracommissural nucleus have been reported in most teleosts investigated (see Wullimann and Northcutt, 1988; Striedter,
1990). The paracommissural nucleus appears to be a conserved cerebellopetal center that acts as a relay in a descending pathway from the telencephalon to the cerebellum (see below; Karten and Finger, 1976; Ito et al., 1982a;
Wullimann and Northcutt, 1988; Striedter, 1990; Imura et
al., 2003). Our previous results in trout showing that the
only conspicuous telencephalic projection to the paracommissural nucleus originates from the central zone of the
dorsal telencephalon (Dc) (Folgueira et al., 2004b), together with the conspicuous cerebellopetal projection of
this nucleus (present results), indicate that the
telencephalo-paracommissural-cerebellar pathways originate from Dc in this species. In turn, Dc receives fibers
from pallial and subpallial regions (Folgueira et al.,
2004b), suggesting that it conveys information from different telencephalic regions. In tilapia, however, both Dc
and the dorsal part (Dd) of the dorsal telencephalon
project to the paracommissural nucleus (Imura et al.,
2003), indicating the existence of species differences in the
origin of this indirect telencephalo-cerebellar projection.
At caudal diencephalic/midbrain levels of trout, there is
a precerebellar nucleus, ventral tegmental nucleus, as
reported in the green sunfish and the goldfish (Wullimann
and Northcutt, 1988). Nevertheless, the number of labeled
cells and the size of the nucleus seem to be greater in trout
than in these species. These cells appear to correspond to
Fig. 15. Schematic representation of the brain in a lateral view
summarizing the afferent (A) and efferent (B) connections of the
valvula cerebelli and corpus cerebelli, and the connections of the
caudal lobe (C) and granular eminences (C). For abbreviations, see
list. [Color figure can be viewed in the online issue, which is available
at www.interscience.wiley.com.]
The Journal of Comparative Neurology. DOI 10.1002/cne
560
the nucleus tegmentocerebellaris and a more lateral precerebellar tegmental group described in advanced teleosts
(Percomorpha) (Uchiyama et al., 1988; Imura et al., 2003).
Precerebellar cells were observed in the dorsal tegmental
nucleus of the green sunfish and the goldfish (Wullimann
and Northcutt, 1988), which may be considered a rostral
part of the lateral nucleus of the valvula receiving telencephalic projections (Yang et al., 2004). In the trout, as in
other teleosts, the lateral nucleus of the valvula mainly
projects to the cerebellum (see below). In chondrosteans
there is no defined lateral nucleus of the valvula, but
numerous precerebellar neurons are located in the midbrain tegmentum; these precerebellar tegmental cell populations might be homologous to the teleost lateral nucleus of the valvula (Huesa et al., 2003).
Wullimann and Northcutt (1988, 1989) described precerebellar cells in the nucleus isthmi of the green sunfish
and the goldfish after HRP application to the corpus and
valvula cerebelli. In trout the nucleus isthmi is well characterized (Pérez et al., 2000); no precerebellar cells were
observed in this nucleus in experiments involving DiI
application to the cerebellum (present results), although
DiI application to the corpus cerebelli labeled cells just
lateral to the nucleus isthmi associated with a different
neuropil, here termed the paraisthmic nucleus. These results in trout appear consistent with results in Navodon
and carp, in which no anterograde labeled fibers observed
in the cerebellum after tracer application to the nucleus
isthmi (Sakamoto et al., 1981; Ito et al., 1982b; Xue et al.,
2001). The partial discrepancy with Wullimann and
Northcutt’s results is probably due to the fact that the
trout nucleus isthmi were defined as the tectum-related
nucleus (Pérez et al., 2000), which does not include the
adjacent precerebellar cells and neuropil. On the other
hand, precerebellar cells were observed in the EdingerWestphal nucleus of the goldfish and the sunfish (Wathey
and Wullimann, 1988; Wullimann and Northcutt, 1988),
although not in the kelp bass (Wathey and Wullimann,
1988) or the trout (present results).
DiI application to the corpus cerebelli of Oncorhynchus
mykiss led to labeling of perikarya in the locus coeruleus,
located in the rostral rhombencephalon. The trout locus
coeruleus consists of a small number of large, multipolar
catecholaminergic perikarya located in the isthmicrhombencephalic region (Manso et al., 1993), as in other
teleosts (Ekström et al., 1986). In chondrosteans, no projection from the locus coeruleus to the cerebellum was
observed (Huesa et al., 2003). At caudal rhombencephalic
levels, DiI application to the corpus cerebelli led to the
labeling of cells in several reticular and octavolateral areas, in agreement with results in other fishes (Finger,
1978a; Meek et al., 1986a; Wullimann and Northcutt,
1988; Fiebig, 1988; Xue et al., 2004). Nevertheless, no
labeled neurons were observed in the raphe or the lateral
cuneate nucleus after DiI application to the corpus cerebelli, unlike in other teleosts (Ito et al., 1982a; Wullimann and Northcutt, 1988; Xue et al., 2004).
The inferior olive is a rhombic lip-derived group of cells
located at the ventromedial surface of the caudal
rhombencephalon. Our results indicate that the trout inferior olive projects to the contralateral cerebellum, as
reported in other teleosts (Finger, 1983; Meek et al.,
1986a,b; Sas and Maler 1987; Wullimann and Northcutt,
1988, 1989), chondrosteans (Huesa et al., 2003), elasmobranchs (Fiebig, 1988), and tetrapods (amphibians:
M. FOLGUEIRA ET AL.
González et al., 1984; reptiles: Bangma and ten Donkelaar, 1982; birds: Arends and Voogh, 1989; mammals:
Brodal et al., 1975; Groenewegen and Voogd, 1977; Brown,
1980; Buisseret-Delmas and Angaut, 1989). The cells of
the inferior olive give rise to cerebellar climbing fibers,
which in teleosts predominantly terminate on cell bodies
and proximal dendrites of Purkinje cells (Pouwels, 1978c;
Finger, 1983). The presence of olivo-cerebellar climbing
fibers appears to be a highly conserved feature of jawed
vertebrates.
The present results show that the cerebellar efferents in
the rainbow trout are mainly contralateral, coursing in
two main pathways (medial and lateral) as reported in
other teleosts (Ito et al., 1982a, 1986; Murakami and
Morita, 1987; Wullimann and Northcutt, 1988, 1989;
Ikenaga et al., 2002; Xue et al., 2004) and chondrosteans
(Huesa et al., 2003). The medial pathway (brachium conjunctivum) projects to the medial midbrain, thalamus, and
pretectum. In the midbrain, cerebellar efferents contact
the nucleus of the medial longitudinal fascicle, as reported
in teleosts (Finger, 1978a; Meek et al., 1986b; Murakami
and Morita, 1987; Wullimann and Northcutt, 1988;
Striedter, 1990; Ikenaga et al., 2002) and other fishes
(chondrosteans: Huesa et al., 2003; elasmobranchs:
Fiebig, 1988). In trout, cerebellar efferents also project to
our ventromedial nucleus of the midbrain reticular formation, a characteristic compact tegmental nucleus. In the
himé salmon, this nucleus has previously been reported to
give rise to a pathway descending to the spinal cord, and
termed “nucleus ruber” (Oka et al., 1986). Our experiments involving DiI application to the spinal cord also
revealed this compact nucleus of rounded medium-sized
cells, like those described by Oka et al. (1986), but we
could assess that its spinal projection was ipsilateral. This
suggests that the “nucleus ruber” of Oka et al. (1986) is
not homologous to the nucleus ruber found in tetrapods,
because in tetrapods the rubrospinal projections are distinctively crossed (ten Donkelaar et al., 1980; LarsonPrior and Cruce, 1992). All reticulospinal cells of the larval zebrafish midbrain also appear to be ipsilateral (see
Gahtan et al., 2002), as found here in rainbow trout. A
small number of cells projecting contralaterally to the
spinal cord and located in the midbrain tegmentum, and
hence corresponding to the tetrapod nucleus ruber, were
described in goldfish (Prasada Rao et al., 1987).
Cerebellar projections to the optic tectum have been
observed in ostariophysans, percomorpha, and mormyrids
(Finger, 1978a; Meek, 1986a; Murakami and Morita,
1987; Ikenaga et al., 2002), but not in the rainbow trout
(present results) and the sturgeon (Huesa et al., 2003).
Cerebellar projections to the torus longitudinalis and hypothalamic areas have been reported in some teleosts (Ito
and Kishida, 1978; Wullimann and Northcutt, 1988;
Striedter, 1990; Ikenaga et al., 2002), but not in the percomorph Sebastiscus marmoratus (Murakami and Morita,
1987). Recent investigations in the carp and four species of
holocentrids (Ito et al., 2003; Xue et al., 2003) indicate that
the cerebellum does not project to the torus longitudinalis,
but fibers labeled in the cerebellum after tracer injection
in torus longitudinalis belong to axons of paracommissural nucleus neurons projecting to the cerebellum. Although labeled fibers were also observed in the torus longitudinalis of trout after DiI application to the cerebellar
corpus and valvula, no labeled perikarya were observed in
the cerebellum after DiI application to the torus longitu-
The Journal of Comparative Neurology. DOI 10.1002/cne
CEREBELLAR CONNECTIONS IN TROUT
dinalis. Likewise, these fibers probably represent collaterals of axons of precerebellar neurons, because a number of
cells were labeled from the torus in the paracommissural
and intermediate pretectal nuclei of trout (present results), which is in line with these recent studies in other
species (Ito et al., 2003; Xue et al., 2003). Therefore, although the areas receiving fibers from the corpus cerebelli
are rather similar in different teleosts (Finger, 1978a;
Murakami an Morita, 1987; Wullimann and Northcutt,
1988; Striedter, 1990; Ikenaga et al., 2002) and chondrosteans (Huesa et al., 2003), minor differences exist. Some
of these differences may reflect methodological constraints
rather than genuine species differences.
Connections of the valvula cerebelli. The valvula
cerebelli of trout receives most afferent projections from
pretectal nuclei (central pretectal, paracommissural, and
intermediate nucleus), the lateral nucleus of the valvula
(LV) (dorsal tegmental nucleus, see above), and the inferior olive, in agreement with results in the goldfish and
sunfish (Wullimann and Northcutt, 1989). Only occasional
labeled cells were observed in the torus semicircularis and
in rhombencephalic reticular regions. In sturgeon, the
neurons afferent to the valvula are located in the pretectum (a few), midbrain tegmentum (a large number; possible LV homolog, see above), torus semicircularis (scarce),
and inferior olive (a few) (Huesa et al., 2003), which is
rather similar to that observed in the trout. In contrast
with the corpus cerebelli, the valvula cerebelli of trout
does not receive fibers from the ventral tegmental nucleus,
the central gray, and the octavolateral area, and does not
project to hypothalamic regions. Other nuclei afferent to
the valvula cerebelli (perilemniscal nucleus, nucleus preeminentialis) reported in the green sunfish and the goldfish (Wullimann and Northcutt, 1989) were not observed
in the rainbow trout (present results). An afferent projection from the isthmic primary sensory trigeminal nucleus
to the valvula cerebelli has been reported in Carassius,
but is not present in Lepomis (Wullimann and Northcutt,
1989) or trout (present results). These authors have related this input to the importance of tactile information
during the complex feeding behavior of cyprinids. The
mormyrids show a hypertrophied valvula cerebelli, most
parts of which are dedicated to the processing of electrosensory stimuli (Nieuwenhuys and Nicholson, 1969a;
Finger et al., 1981), so it is difficult to compare their
valvular connections (Finger et al., 1981) with those of
nonelectrosensory teleosts. In any case, the mormyrid valvula receives many of its afferents from nuclei that are
also prevalvular in other teleosts (pretectum, nucleus lateralis valvulae) (Finger et al., 1981; Meek et al., 1986a).
The valvular peduncle of the mormyrid cerebellum additionally appears to receive olivo-cerebellar projections
(Meek et al., 1986a). Further comparison with these electric fishes is beyond the scope of this article.
Connections of the caudal lobe. After DiI application
to the caudal lobe, only a few labeled cells were observed
in the lateral nucleus of the valvula and in the paracommissural nucleus, suggesting that these nuclei are not the
most important afferents to this cerebellar lobe. DiI application to the caudal lobe led to labeling of fibers extending
to the level of the obex, not observed with other types of
DiI application. Although the origin of these fibers could
not be ascertained, they may be afferents from the lateral
cuneate nucleus or spinal cord. Fibers from these origins
have been observed in other teleosts after HRP applica-
561
tion to the corpus (Ito et al., 1982a; Murakami and Ito,
1985; Wullimann and Northcutt, 1988).
Retrogradely labeled eurydendroid cells were observed
in the caudal lobe of the rainbow trout after DiI application to various brain regions (central pretectal nucleus,
paracommissural nucleus, ventral tegmental nucleus, and
nucleus of the medial longitudinal fascicle). In Sebastiscus
marmoratus, Murakami and Morita (1987) showed that
caudal lobe eurydendroid cells project to the ventromedial
thalamus, oculomotor region, and the nucleus ruber.
Connections of the granular eminences. After DiI
application to the granular eminences, retrogradely labeled cells were observed mostly in the lateral nucleus of
the valvula and also at rhombencephalic levels (superior
reticular nucleus, locus coeruleus, medial octavolateralis
nucleus, and lateral reticular region), but not in the pretectum or inferior olive. This indicates on the one hand
that this granular layer receives most projections (mossy
fibers) from the LV, lacking olivocerebellar climbing fibers, and on the other hand that this region receives
rather selectively crossed fibers from two reticular regions
(superior reticular nucleus and lateral reticular nucleus).
In view of their location in regions of fiber passage, they
appear to send to the cerebellum inputs from multiple
origins. In chondrosteans, the granular eminences also
receive most projections from the precerebellar midbrain
tegmentum (Huesa et al., 2003), i.e., from the possible LV
homolog.
Primary sensory fibers labeled from the trout granular
eminence were observed in the anterior lateral line nerve
and likely in the posterior lateral line nerve. Lateral line
nerve projections terminating in the granular eminence
have been observed in various teleosts including the rainbow trout (Schellart et al., 1992; see Meek and Nieuwenhuys, 1998, for further details), in holosteans (Song and
Northcutt, 1991) and in chondrosteans (New and Northcutt, 1984; Huesa et al., 2003).
On the other hand, DiI application to the granular eminences led to labeling of varicose parallel fibers reaching
the molecular layer of the caudal lobe and the cerebellar
crest of the octavolateral region. Efferents from granule
cells of the granular eminences to the cerebellar crest and
caudal lobe molecular layer have also been reported in
various other teleosts, including electroreceptive species
(Bass, 1982; Meek et al., 1992b; see also Meek and Nieuwenhuys, 1998), and in chondrosteans (Huesa et al.,
2003).
Connections of the lateral nucleus of the valvula.
The lateral nucleus of the valvula is probably the precerebellar nucleus with most numerous neurons in the rainbow trout, so knowledge of its afferents is important for
understanding cerebellar circuitry. In two experiments,
the LV was approached in sectioned brains from the caudal end, so only cells rostral to the section plane could be
studied. Cells afferent to the trout LV were observed in
the telencephalon (ventral nucleus of the ventral area;
Folgueira et al., 2004a,b), in the preoptic nucleus, in nuclei of the pretectal region (central pretectal, paracommissural nucleus, intermediate pretectal, ventral accessory
optic nucleus), in the central posterior thalamic nucleus
(occasional cells), in the posterior tubercle nucleus, and in
the diffuse nucleus of the inferior hypothalamic lobe
(present results). A recent study of tilapia has also revealed projections to the LV from the dorsal telencephalon, habenula, dorsomedial thalamus, pretectum, mam-
The Journal of Comparative Neurology. DOI 10.1002/cne
562
millary body, central nucleus of the inferior lobe, anterior
and posterior tuberal nuclei, torus semicircularis, dorsal
horn, and lateral funicular nucleus (Yang et al., 2004),
which show only partial resemblance to those observed by
us in the trout. In the carp, labeled cells from the LV were
observed in the diffuse nucleus and nucleus ventromedialis thalami (Ito and Yoshimoto, 1990). On the other hand,
in trout no labeled cells were observed in the magnocellular superficial pretectal nucleus (PSM), which projects to
the LV in the carp and goldfish (see Northcutt and
Braford, 1984; Ito et al., 1997). Although there is the
possibility that the nuclei termed PSM in trout and carp
were not homologous, available results suggest that diencephalic inputs to LV vary widely among teleost species,
perhaps reflecting divergent specialization of forebrain
centers. Although the region of the LV of goldfish receiving telencephalic afferents has been considered by some
authors as a different nucleus termed the dorsal tegmental nucleus (Wullimann and Northcutt, 1988, 1989), other
authors consider it an LV region (Yang et al., 2004;
present results).
Retrogradely labeled cells were observed in the lateral
nucleus of the valvula after DiI application to all regions of
the trout cerebellum. As far as we know, the efferent
projection from the LV to the cerebellum has been observed in all teleost species studied (Ito et al., 1982a;
Finger, 1983; Meek et al., 1986a,b; Wullimann and Northcutt, 1988, 1989; Striedter, 1990; Ito and Yoshimoto, 1990;
Imura et al., 2002; Xue et al., 2004). Topographical organization of the projections from the LV to the valvula
cerebelli and corpus cerebelli has been reported in several
teleosts (Finger, 1978a; Meek et al., 1986b; Wullimann
and Northcutt, 1989; Ito and Yoshimoto, 1990). Although
in trout the valvula appears to receive fibers mainly from
medial regions of the LV, further investigation is necessary to confirm whether the LV-cerebellar projections are
topographically organized.
Eurydendroid cells of trout. Tracer application to
various brain areas (the central pretectal nucleus, the
paracommissural nucleus, the ventral tegmental nucleus,
and the nucleus of the medial longitudinal fascicle) led to
the labeling of efferent neurons of the cerebellum, termed
eurydendroid cells (Nieuwenhuys and Nicholson, 1969b;
Finger, 1978b; Pouwels, 1978b). The best staining of the
eurydendroid cells was obtained after DiI application to
the nucleus of the medial longitudinal fascicle, with long
incubation periods. Labeled eurydendroid cells were
mainly distributed in the valvula cerebelli and the caudal
lobe. Two different morphologies of eurydendroid cells
were identified within the trout cerebellum, fusiform and
multipolar, probably corresponding respectively to the
types A and B reported in Sebastiscus marmoratus (Murakami and Morita, 1987), although the type A cell possess a single dendrite. Three types of eurydendroid cells,
fusiform, multipolar (polygonal), and pear-shaped (monopolar), have been recently described in the goldfish cerebellum (Ikenaga et al., 2005). As stated by other authors
(Nieuwenhuys and Nicholson, 1969a; Nieuwenhuys et al.,
1974; Pouwels, 1978b; Finger, 1978b), trout eurydendroid
cells project to a number of nuclei in the brain.
In the present study some monopolar cells with dendrites ascending in the molecular layer were labeled in the
caudal lobe after performing tract-tracing reciprocal experiments with tracer application in the medial octavolateralis nucleus. Our results indicate that some cells of
M. FOLGUEIRA ET AL.
this caudal cerebellar region may project directly to the
octavolateralis region. These cells appear to be eurydendroid cells, although confirmation must await further investigation with molecular markers of Purkinje cells. Similar results were obtained in sturgeon after DiI application
to the octavolateral region (Huesa et al., 2003). The existence of direct projections from the vestibulocerebellum
toward octavolateral centers originating from Purkinje
cell has been reported in other vertebrates (Dietrichs et
al., 1983; Epema et al., 1985; Arends and Zeigler, 1991).
Telencephalo-cerebellar pathways. In various teleost fishes the telencephalon maintains indirect connections with the corpus cerebelli via 1) the paracommissural
nucleus, 2) the dorsal preglomerular nucleus, and 3) the
dorsal tegmental nucleus/LV (see Wullimann and Meyer,
1993; Imura et al., 2003; Yang et al., 2004; present results). Other pathways can also exist (Imura et al., 2003).
1) The indirect telencephalo-cerebellar pathway via the
paracommissural nucleus has been described in various
species of euteleosts (Karten and Finger, 1976; Ito et al.,
1982a; Wullimann and Northcutt, 1988; Striedter, 1990;
Imura et al., 2003). In the rainbow trout, the paracommissural nucleus can also be identified by its position and
hodology (Folgueira et al., 2004b; present results). This
nucleus receives afferent fibers from the central area of
the dorsal telencephalon (Dc) and gives rise to a conspicuous tract coursing caudally to the lateral nucleus of the
valvula and to the cerebellum (corpus and valvula)
(Folgueira et al., 2004b; present results). These connections are different from those reported in the cichlid Oreochromis niloticus (Imura et al., 2003; Yang et al., 2004), in
which the paracommissural nucleus lacks connection with
the LV and receives projections from both the central and
dorsal areas of the dorsal telencephalon (Dd).
2) In osteoglossomorphs, the dorsal preglomerular nucleus is an indirect relay center in the telencephalic pathway to the cerebellum (Meek et al., 1986a,b; Wullimann
and Northcutt, 1990; Wullimann and Meyer, 1993). In the
rainbow trout, the preglomerular nuclei and mammillary
body complex maintain connections with several telencephalic areas (Folgueira et al., 2004a,b, 2005), but no efferent projections to any precerebellar nuclei were observed.
These results in trout are in partial agreement with those
reported in the ostariophysan Ictalurus punctatus (Striedter 1990), in which anterograde labeled fibers but no retrograde labeled cells were observed in the preglomerular
complex after DiI application to the corpus cerebelli. This
complex does not seem to form part of any descending
telencephalo-cerebellar pathway, either in trout or I.
punctatus. The telencephalo-cerebellar pathway via the
preglomerular nucleus has been proposed to be apomorphic in the osteoglossomorph line (Wullimann and Meyer,
1993). An alternative explanation (Xue et al., 2003) suggests that the dorsal preglomerular nucleus of osteoglossomorphs is homologous to the paracommissural nucleus
of other teleosts, because it receives fibers from Dc and
projects to the torus longitudinalis and cerebellum, as
does the paracommissural nucleus. In this case, the osteoglossomorph novelty would consist of the migration of the
paracommissural nucleus far away from the location typical in other teleosts.
3) A dorsal tegmental nucleus adjacent to the LV has
been identified in various nonelectroreceptive teleosts
(Vanegas and Ebbesson, 1976; Murakami et al., 1983;
Wullimann and Northcutt, 1988, 1989, 1990; Wullimann
The Journal of Comparative Neurology. DOI 10.1002/cne
CEREBELLAR CONNECTIONS IN TROUT
and Meyer, 1993) as a relay nucleus in the pathway carrying information from the telencephalon to the cerebellum. The cerebellopetal character of the “dorsal tegmental
nucleus” has also been reported in electroreceptive fishes
(Meek et al., 1986a; Wullimann and Northcutt, 1990).
After DiI application to various telencephalic areas of the
trout (Dc, Dp, Vv; see Folgueira et al., 2004a,b, for further
details), labeled fibers were observed in a lateral prominence of the LV, but this part is cytoarchitectonically
indistinguishable from the remainder of the LV, as also
found in tilapia (Yang et al., 2004). Our reciprocal experiments could not unequivocally confirm the origin of these
fibers in the ventral nucleus of the ventral telencephalic
area (Folgueira et al., 2004a). Accordingly, two of these
indirect telencephalo-cerebellar pathways were observed
in trout: the telencephalo-paracommissural-cerebellar,
and the telencephalo-LV-cerebellar pathways.
In addition to the above-mentioned pathways, another
indirect telencephalo-cerebellar pathway may be present
in trout. Our previous results reveal that the central gray
receives telencephalic afferents (Folgueira et al., 2004a,b).
In turn, cells of this region project to the corpus cerebelli
(present results), so these precerebellar neurons could be
relaying descending telencephalic inputs to the cerebellum.
Finally, although a direct cerebello-telencephalic pathway projecting to the pallium (Dc) has been described in
electroreceptive teleosts (Wullimann and Rooney, 1990)
and in goldfish (Vonderschen et al., 2001), no indication of
the presence of such a projection was obtained in tracttracing studies of trout (Folgueira et al., 2004a,b; present
results).
ACKNOWLEDGMENT
We thank Mrs. Pilar Gómez (Piscifactorı́a Berxa, Mesı́a,
A Coruña) for supplying the biological material used in
this study.
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