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Afferent and efferent connections of the habenula in the rainbow trout (Oncorhynchus mykiss) An indocarbocyanine dye (DiI) study

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Afferent and Efferent Connections
of the Habenula in the Rainbow Trout
(Oncorhynchus mykiss):
An Indocarbocyanine Dye (DiI) Study
Department of Cell and Molecular Biology, University of La Coruna, 15071 La Coruna, Spain
(J.Y.); Department of Fundamental Biology, University of Santiago de Compostela,
15706 Santiago de Compostela, Spain (R.A.)
The habenula is a conserved structure in the brain of vertebrates. With the aim of further
understanding of the evolution of the habenular system in vertebrates, we studied the afferent
and efferent connections of the habenula of the rainbow trout. Experiments included
application of the carbocyanine dye l,l’-dioctadecyl-3,3,3’,3’-tetramethylindoc~bocyanine
perchlorate (DiI) into the habenula, telencephalon, pineal organ, posterior tubercle, and
interpeduncular nucleus (IPN). The results obtained reveal a consistent pattern of habenular
connections. Most afferents originate from three nuclei, one extending from the preoptic region
to the rostral thalamus (the entopeduncular nucleus), the second located in the region of the
hypothalamus-posterior tubercle and consisting of large bipolar cells (tuberculohabenular
nucleus), and the third in the preoptic region (preoptic nucleus). A few large neurons of the
locus coeruleus appeared to be labeled in some cases. The trout habenula also receives pineal
and parapineal projections. Small labeled glial cells were observed in the thalamus around the
fasciculus retroflexus and, sometimes, around the IPN. The most conspicuous efferents coursed
in the fasciculus retroflexus to the IPN, the isthmal raphe, and the central gray. The existence
of olfactohabenular or habenulotelencephalic projections is discussed. o 1996 Wiley-Liss, Inc.
Indexing terms: limbic system, carbocyanine dye, fasciculus retroflexus, entopeduncular nucleus,
The habenula is an evolutionarily conserved structure in
the brain of vertebrates. It is located in the caudal epithalamus (synencephalon), and, in mammals, it consists of two
habenular nuclei, the medial and the lateral (Herkenham
and Nauta, 1977). A similar distinction between the medial
and lateral habenular nuclei is found in some nonmammalian vertebrates (Distel and Ebbesson, 1981; Diaz and
Puelles, 1992b). The habenular nuclei are connected to
several telencephalic, diencephalic, and mesencephalic centers. In mammals, afferent fibers to the habenula arise from
telencephalic regions (entopeduncular nucleus, septum,
nuclei of the diagonal band, substantia innominata) and
from diencephalic and mesencephalic nuclei (Herkenham
and Nauta, 1977; Parent et al., 1981).The efferents of the
habenula course in the fasciculus retroflexus (FR),which is
also called the tractus habenulointerpeduncularis, and
project to the interpeduncular nucleus (IPN) and to several
regions of the mesencephalon, diencephalon and telencephalon (Cajal, 1911; Herkenham and Nauta, 1979; Contestabile and Flumerfelt, 1981). There have also been a few
experimental studies on habenular connections in nonmammals. Tracing techniques have indicated that the habenular
connections of frog (Kemali et al., 1980; Kemali and Lazar,
1985) and lizard (Distel and Ebbesson, 1981; Diaz and
Puelles, 1992a,b), in many respects, are similar to those of
mammals. However, frog habenular afferents originate
exclusively in nuclei from the telodiencephalic boundary
(Kemali et al., 1980). In the larval lamprey, the pattern of
habenular connections is even simpler; afferents originate
mainly from the lobus subhippocampalis, and efferents
course in the FR to the neuropil of the IPN (Yafiez and
Anadon, 1994).
Classical studies in teleosts with nonexperimental methods (Sheldon, 1912; Holmgren, 1920) indicated that the
habenula receives fibers from the ventromedial and ventroAccepted April 2,1996.
Address reprint requests to Dr. R. Anadh, Department of Fundamental
Biology, Faculty of Biology, University of Santiago de Compostela, Santiago
de Compostela 15706, Spain. E-mail:
J. Y ~ E AND
lateral parts of the telencephalon through the medial and
lateral olfactohabenular tracts, which, together, form the
stria medullaris. According to Holmgren (19201, the habenula receives fibers from the diencephalon (nucleus
rotundus) through the tractus diencephalohabenularis. This
author also mentions a tractus eminentia-habenularis,
which originates in cells of the middle part of the eminentia
thalami (i.e., regio posthabenularis). In addition, the teleost
habenula receives fibers from the pineal and parapineal
organs (Holmgren, 1920; Rudeberg, 1969; Ekstrom and van
Veen, 1984). In the goldfish, Sheldon (1912) describes other
afferents to the habenula from the hippocampus (i.e., dorsal
telencephalon), the nucleus teniae (i.e., nucleus interpeduncularis), three parts of the nucleus preopticus, the thalamic
tubercle (“Haubenwulst”), and the diencephalon. The habenular efferents run mainly in two bundles, the tractus
habenulothalamicus and the tractus habenulopeduncularis
or fasciculus retroflexus (Holmgren, 1920; Kappers et al.,
1936; Villani et al., 1987, 1994a,b).Strikingly, and despite
the numerous telencephalohabenular projections reported
on the basis of classical techniques (Sheldon, 1912;
Holmgren, 1920; Kappers et al., 1936),there are no modern
tracing studies of such projections. However, the results of
total telencephalic ablation and bulk telencephalic labeling
(Striedter, 1990; Villani et al., 1994a) indicate that telencephalohabenular connections exist. The neurons of origin
of these telencephalic connections are currently unknown.
Although, in some species, secondary olfactory fibers cross
in the habenular commissure, they do not contribute
significantly to telencephalohabenular projections (Finger,
1975; Bass, 1981b; von Bartheld et al., 1984; Prasada Rao
and Finger, 1984; Levine and Dethier, 1985; Sas et al.,
1993). Furthermore, no diencephalic or mesencephalic
nucleus, apart from the IPN (Villani et al., 1987, 1994a,b),
has yet been shown experimentally to be connected with the
teleost habenula. In the present study, we conducted
experiments with the indocarbocyanine dye 1,l‘-dioctadecyl3,3,3’,3‘-tetramethylindocarbocyanine
perchlorate (DiI),
which diffuses in plasma membranes of fixed tissues, to
determine both the origin of habenular afferents and the
targets of habenular efferents in a teleost, the rainbow
trout (Oncorhynchus mykiss, Salmoniformes).
Forty rainbow trout, which were obtained from a local
fish farm, were used in the experiments. Trout were deeply
anaesthetized with tricaine methane sulphonate (MS-222;
Sigma) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were carefully
dissected out of the skull and were then embedded in 3%
agarose. Crystals of DiI (Eugene, OR) were then applied to
the structures under investigation following a modification
of Holmqvist et al.’s (1992) method. In brief, blocks were
cut at the level of the habenula (or other structures) with a
razor blade, crystals of DiI were applied, and the surface
was covered with agarose. The brains were then left in
fixative for 7-60 days at 37°C. After the transport period,
transverse or sagittal sections (50-100 pm thick) obtained
by using a Vibratome were examined and photographed
with a Nikon fluorescence photomicroscope equipped with a
rhodamine filter set and with Nomarski differential interference contrast (DIC).
Three types of block were used: 1) with the habenula
connected to the brain caudal to it, 2) with the habenula
connected to the ipsilateral telencephalic lobe and to the
caudal brain, and 3) with the habenula connected to the
entire forebrain. In addition, DiI was applied to several
other regions (interpeduncular neuropil, isthmal central
gray, posterior tubercle, entopeduncular nucleus, and pineal organ) to confirm their habenular connections.
Series of Nissl-stained transverse sections of rainbow
trout brains from collections in our laboratory were also
used for topographic purposes. The nomenclature for regions and nuclei of the trout brainstem was adapted from
Nieuwenhuys and Pouwels (1983), that for the diencephalon was adapted from Holmgren (1920) and from Bergqvist
(19321, and that for the telencephalon was adapted from
Northcutt and Braford (1980).
The habenulae of the rainbow trout are similar in
appearance, although the right habenula is somewhat
larger. Unlike in other actinopterygians (Braford and Northcutt, 1983),there is no clear distinction between dorsal and
ventral nuclei in trout (Holmgren, 1920).
Application of DiI to the habenula led to intense labeling
of the habenular (i.e., superior) commissure and a few fiber
tracts (fasciculus retroflexus, habenulothalamic tract, tuberculohabenular tract, and stria medullaris). Labeling was
most prominent on the side ipsilateral to the application
site (Fig. l),but the contralateral fasciculus retroflexus,
dorsolateral thalamic area
pretectal area
ventrolateral thalamic area
anterior commissure
horizontal commissure
posterior commissure
dorsocentral telencephalic area
dorsolateral telencephalic area
dorsomedial telencephalic area
fasciculus retroflexus
hypothalamic lobe
interpeduncular nucleus
trochlear motor nucleus
locus coeruleus
nucleus diffusus hypothalami
entopeduncular nucleus
nucleus pseudoglomerulosus
nucleus lateralis valvulae
nucleus preglomerulosus
preoptic nucleus
nucleus sacci vasculosi
nucleus anterior tuberis
tuberculohabenular nucleus
optic tectum
optic chiasma
saccus vasculosus
torus lateralis hypothalami
mesencephalic tegmentum
torus semicircularis
valvula cerebelli
ventrolateral telencephalic area
ventromedial telencephalic area
Fig. 1. Chartings of transverse sections of the trout brain showing nine perchlorate (DiI) application to the habenula (shaded area). For
the ipsilateral distribution of the habenular afferent neurons (small the contralateral connections, see the text. The fasciculus retroflexus is
circles) and the fiber tracts (dashes) and fields of terminal fibers (dotted indicated by a large dot. The inset indicates the levels of the sections.
For abbreviations, see list. Scale bar = 1mm.
areas) revealed after l,l’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocya-
Fig. 2. Fluorescence micrograph of section through the entopeduncular nucleus after DiI application to the habenula. This sagittal section
passing through the labeled habenula (asterisk) shows the organization
of the entopeduncular afferents to the habenula (thin arrow) and the
origin of the fasciculus retroflexus (wide arrow). Star, entopeduncular
nucleus. The orientation of the figure is indicated by the perpendicular
arrows (d, dorsal; r, rostral). Scale bar = 250 pm.
Fig. 3. Fluorescence micrograph of section through the entopeduncular nucleus after DiI application to the habenula. Transverse section
through the ipsilateral entopeduncular nucleus showing a bundle of
habenulopetal fibers (arrow).Note the highly fluorescent central region
of the nucleus. Scale bar = 100 p m .
tuberculohabenular tract, and stria medullaris were also
the habenula, with many small bundles converging at this
point (Fig. 2). In these sections, the origin of the fasciculus
retroflexus from several bundles is also evident, although,
on the ipsilateral side, these bundles were massively labeled, and it was difficult to observe details.
One result of DiI application to the habenula was the
labeling of numerous fibers in two conspicuous telencephalic tracts, the corticohabenular (lateral) and olfactohabenular (medial) tracts. The lateral fibers ran between the
dorsal and ventral telencephalic areas near the meningeal
surface, in the ventrolateral region rich in somatostatinimmunoreactive neurons described by Becerra et al. (1995;
nucleus olfactorius lateralis of Sheldon, 1912; lateral nucleus
of the area ventralis of Bass, 1981a). Medial fibers ran in
the middle of the area ventralis (nucleus olfactorius medialis of Sheldon, 1912). Very interestingly, these fibers give
rise to highly conspicuous terminal fields (Figs. 6, 7). Some
fibers running in the olfactohabenular tract were found to
end in the rostral telencephalon, and a few reached the
ventral portion of the olfactory bulb (Fig. 8). No labeled
neurons were observed at the end of these tracts, whereas
the labeled fibers were observed to branch and give rise to
terminal fields; thus, these fibers are clearly telencephalic
afferents. The origin of these tracts can be traced to the
entopeduncular nucleus, from which three main radiations
of fibers appear to arise (rostrolateral, rostromedial, and
dorsomedial). In sagittal sections, individual nerve fibers
were observed to branch. Very probably, these telencephalic
afferents represent collaterals of habenular afferents originated from the entopeduncular nucleus, and not habenular
Afferent neurons
Neurons giving rise to habenulopetal projections were
present in a few areas of the ventral telencephalon, diencephalon, and mesencephalon (Fig. 1).Interestingly, only a
few cells were labeled in the telencephalon rostral to the
preoptic region. Following Holmgren (19201, we include
this region, which extends around the preoptic recess from
caudal to the anterior commissure, to the optic chiasma, as
part of the telencephalon.
Very numerous afferent cells were located ipsilateral to
the application site in a long nucleus extending from the
preoptic region caudal, to the anterior commissure, to the
rostral thalamus (Figs. 2, 3), accompanying the largest
telodiencephalic tract (lateral forebrain bundle). These
neurons correspond to the entopeduncular nucleus (area
somatica of Holmgren, 1920). The nucleus was so heavily
labeled and its neurons were so densely packed that observation of the shape of individual neurons was generally not
possible on the ipsilateral side. Neurons were also labeled
contralaterally to the application site, although more
sparsely. Here, labeled neurons of the entopeduncular
nucleus show a round perikaryon and sparsely branched
long dendrites (Fig. 41, which extend in several directions,
including ventral, medial, rostral, and caudal. The dendrites of these neurons bear small spines in some areas (Fig.
5). In sagittal sections, the course of the axons of these
entopeduncular cells was characteristic: They are grouped
in small bundles, which run first caudally then, at the level
of the habenula, turn dorsally or rostrodorsally to ascend to
Figs. 4-7. Labeled cells and processes in the contralateral (Figs. 4,
5) and ipsilateral (Figs. 6, 7) telencephalon after DiI application to the
Fig. 4. Entopeduncular neuron showing the branching of a thick
dendrite (arrow). Photomontage of a sagittal section. Scale bar =
25 Fm.
Fig. 5 . Detail of a neuronal process of the entopeduncular nucleus
showing short lateral spines (small arrows). Scale bar = 25 Fm.
Fig. 6. Transverse section through the lateral region of the telencephalic lobe showing the labeled “lateral olfactohabenular tract.” Note
the areas of terminal fibers (arrow) and the absence of labeled cells.
Star, meninges. Scale bar = 100 pm.
Fig. 7. Transverse section through the ventral telencephalic area at
the level of the anterior commissure (CAI showing intensely labeled
processes in the infracommissural (star) and supracommissural (asterisk) regions. Note the absence of labeled neurons. Scale bar = 100 pm.
Fig. 8. Detail of terminal fibers in the caudal olfactory bulb. Scale
bar = 50 um.
Fig, 9. Transverse section through the telencephalon ipsilateral to
the DiI labeled habenula. This section through the preoptic nucleus
(asterisk) shows three DiI-labeled cells. Note processes of entopeduncular neurons (upper left). Arrow, lateral optic recess; star, preoptic
recess. Scale bar = 100 um.
application to the ipsilateral hahenula showing the tuberculohabenular
nucleus. Section showing labeled cells extending towards the medial
hypothalamic lobe (star). Asterisk indicates the lateral hypothalamic
lobe. The orientation of the figure is indicated by the perpendicular
arrows (d, dorsal; r, rostral). Scale bar = 250 pm.
Fig. 10. Sagittal section through the posterior tubercle after DiI
Fig. 11. Sagittal section through the posterior tubercle after DiI
application to the ipsilateral habenula showing the tuherculohahenular
nucleus. Detail of the labeled cells of Figure 10. Scale bar = 100 pm.
In the telencephalon, labeled neurons were also observed
in caudal regions but only occasionally rostral to the
anterior commissure. A few pear-shaped cells were observed in the supracommissural region, dorsal to the conspicuous central area ventralis ventralis (Vv) terminal field
(see below). Very few small cells were labeled ventral or
lateral to the anterior commissure or in the central Vv
region, and most of these appeared to be putative oligodendrocytes (see below). In the preoptic region, sparse neurons
were observed along the preoptic nucleus (Fig. 91, both
scattered alongside the lateral area and in the layers of
periventricular neurons lateral and ventral to the magnocel-
lular portion. These neurons are medium sized and are
clearly smaller than the cells of the magnocellular portion.
In general, fibers are scarce in the preoptic region compared
with other regions of the telencephalon. No precommissural
telencephalic neurons were observed in our experiments either
in ventral or dorsal telencephalic areas or in the olfactory bulb.
Following DiI application to the habenula, a large and
conspicuous habenulopetal nucleus can be observed in the
hypothalamus extending from levels lateral to the organon
vasculosum hypothalami to the posterior tubercle, with
some neurons even reaching the dorsal walls of the median
hypothalamic lobe (Figs. 1,10,11). These cells are recognizable in Nissl-stained sections by their scattered distribution
and their spindle-like or piriform shape; they are about 8
km in diameter, somewhat larger than the surrounding
periventricular neurons. With DiI labeling, these cells have
an elegant shape, with very long, sparsely branched dendritic processes (Fig. 11) that follow ventral, dorsal, or
longitudinal courses. These cells lie medial to the preglomerular-pseudoglomerular-mammillary complex of trout,
which, from rostral to caudal, shifts progressively medialwards (Fig. 1). The dendrites of labeled neurons form
conspicuous dendritic fields, which extend over a considerable area. In longitudinal sections, many of these cells
extend in rostrocaudal directions parallel to the major
telodiencephalic bundles. In view of its habenular connection, which, to our knowledge, has not been reported
previously, we denominate this nucleus the “tuberculohabenular nucleus.” Its axons run rostrally and dorsally,
forming a conspicuous tuberculohabenular tract near the
medial wall of the ventricle (Fig. 1); this tract ascends
towards the habenula medially to the tract of the horizontal
The observation that the tuberculohabenular neurons
project to the habenula and not to other related nuclei was
confirmed by application of DiI to the tuberal hypothalamus containing them. This experiment revealed a labeled
tract ascending parallel to the ventricle and conspicuous
fields of terminal fibers in the habenular neuropil (Fig. 12).
A portion of these fibers cross in the habenular commissure; thus, the projection is bilateral, which is in agreement
with the labeling of a few contralateral cells after DiI
application to the habenula.
In the rostral rhombencephalon, a few (one to three)
large labeled neurons were sometimes observed in the locus
coeruleus (Fig. 13). These were multipolar cells with thick
Following DiI application to the habenula, the posthabenular region was generally observed to contain a sparse
population of small round cells medial to the fasciculus
retroflexus, in which processes were hardly distinguishable.
Similar labeled cells were likewise observed in the forebrain
near the medial olfactory tract in the region of the IPN and,
very occasionally, in other areas. These cells appeared to be
oligodendrocytes. A few tanycytes along the posthabenular
course of the fasciculus retroflexus and in the ependymal
walls near the IPN were also stained.
The application of DiI into the habenula led to the
labeling of fibers and cells in the pineal and parapineal
organs. The application of DiI to the pineal vesicle led to the
labeling of pineal fibers; most of these fibers coursed to the
posterior commissure, although some coursed to the habenular commissure. A small subset of these fibers entered
the habenula and seemed to form a plexus at its center.
Pineal fibers also ran to the pretectal area and to the dorsal
and ventral thalamus. Some pineal fibers ran in the fasciculus retroflexus, and a few could be followed to the neuropil
of the IPN.
Application of DiI to the habenula, as noted above, led to
labeling of telencephalic fibers that appeared to be collaterals of habenular afferents originated from the entopeduncular nucleus. To confirm this interpretation, DiI was applied
directly to the entopeduncular nucleus. Such application
led to labeling of fibers in the stria terminalis and the
habenular commissure and labeling of conspicuous terminal fields, but not neurons, in the habenulae. Some entopeduncular nucleus fibers ran in the outer layers of the
fasciculus retroflexus to the dorsomedial thalamus, where
they gave rise to branches and beaded processes; a few of
them even reached the IPN.
Efferent connections
The habenulointerpeduncular system was intensely
stained after DiI application into the habenula. Most labeled efferent fibers of the habenula were found running in
the FR, which could be followed caudoventrally in the
direction of the IPN. This fascicle originates from the union
of several small bundles that converge caudal to the habenula (Fig. 2). The FR forms a compact tract along its
posthabenular course, reaching the ventral tegmentum
near the oculomotor nucleus and running caudally to the
IPN (Fig. 14). The IPN consists of dorsal and ventral
neuropils, separated by glia and bands of fibers. Both the
dorsal and ventral neuropils of the IPN were intensely
labeled (Fig. 15). In the ventral neuropil, individual labeled
fibers could generally be distinguished, whereas, in the
dorsal neuropil, labeling was sometimes so intense that
individual fibers could not be observed (Fig. 15). In cases of
faint habenular labeling, FR fibers were observed to branch
and course transversal or in other directions in the IPN.
Caudal to the IPN, labeled fibers were followed to the
raphe, where they formed a dense neuropil (Fig. 16) from
which some fibers extended caudally and dorsally to the
central gray (Fig. 17) a t isthmal (pretrigeminal) levels. No
labeled neurons were found along the course of the FR,
indicating that this tract is formed of efferent fibers. The
few cells that appeared to be associated with the FR and the
IPN could be interpreted as oligodendrocytes (see below).
Application of DiI to the IPN or to the fasciculus retroflexus at the level of the caudal thalamus provided confirmation of our results with regard to habenulointerpeduncular
projections. Both types of application led to labeling of cells
in the habenulae (Figs. 18, 19). Labeled neurons were
located throughout the nucleus, indicating that all habenular regions give rise to FR fibers. Habenular neurons were
small piriform or stellate cells, with labeled dendrites and
processes that formed part of conspicuous neuropil areas
surrounded by cords of densely packed cells. In addition to
these habenular cells, these types of application led to
labeling of a few large neurons in the perihabenular region
caudal and lateral to the habenula; these cells had long,
sparsely branched processes some with very conspicuous
In addition to the fibers running in the FR and the
isthmal raphe, application of DiI to the habenula led to
labeling of fibers in the telencephalon, diencephalon, and
mesencephalon. In the telencephalon, fibers were observed
in two tracts: lateral and medial. These tracts seem to be
made up of axon collaterals of entopeduncular neurons (see
above), Caudally, labeled fibers were observed to run to the
Fig. 12. Detail of the terminal field of tuberculohabenular fibers in
the habenula after application of DiI to the posterior tubercle. Star,
third ventricle. Scale bar = 50 pm.
trance of the fascicle (black star) in the interpeduncular nucleus. Note
bilaterality of the interpeduncular neuropil. Scale bar = 250 pm.
Fig. 13. Transverse section through the locus coeruleus after DiI
application to the hahenula showing a large labeled neuron. Small
arrows point to the cell axon. Scale bar = 100 pm.
Fig. 14. The fasciculus retroflexus and its terminal fields in the
interpeduncular nucleus after DiI application to the habenula. En-
Fig. 15. The fasciculus retroflexus and its terminal fields in the
interpeduncular nucleus after DiI application to the habenula. The
interpeduncular nucleus at a middle level showing its dorsal and
ventral neuropils. In this case, the high intensity of fluorescence in the
dorsal neuropil obscures individual fibers. Arrows indicate the midline.
Scale bar = 100 pm.
posthabenular dorsomedial thalamus, where they formed a
conspicuous terminal field, and to terminal fields in the
pretectal area at the level of the posterior commissure and
the dorsal tegmentum. Because this type of application
labels the pineal tract, and because DiI application to the
pineal vesicle (see above) led to labeling of pineal fibers that
Fig. 16. Habenular projections to the isthmal raphe and central
gray revealed after DiI application to the habenula. Dense field of
terminals in the raphe (star) just caudal to the interpeduncular
nucleus. Scale bar = 100 mm.
Fig. 17. Habenular projections to the isthmal raphe and central
gray revealed after DiI application to the habenula. Labeled habenular
fibers ascending through the raphe to the isthmal central gray.
Asterisk, aqueduct; stars, medial longitudinal fascicle. Scale bar =
100 km.
Fig. 18. Labeling of the habenula and fasciculus retroflexus after
DiI application to the interpeduncular nucleus. Transverse section
through the habenulae (stars) showing the bilateral labeling of the
fasciculus retroflexus, which, at this caudal level, is formed of many
small bundles (wide arrows). Asterisk, optic tectum. Scale bar =
250 Fm.
Fig. 19. Labeling of the habenula and fasciculus retroflexus after
DiI application to the interpeduncular nucleus. Detail of the left
habenula of Figure 18 showing labeled habenular neurons. Scale bar =
50 km.
form conspicuous terminal fields in these areas, it seems
very probable that these projections were not habenular.
context, because most habenular connections in trout are
Subdivision of the habenulae
To our knowledge, this is the first experimental study of
the afferent and efferent connections of the trout habenula.
Our results show, first, that most habenular afferents arise
from only a few nuclei (notably, the entopeduncular nucleus,
the parvicellular preoptic nucleus, and a habenulopetal
nucleus of the posterior tubercle) and, second, that the
fasciculus retroflexus contains most, if not all, of the
habenular efferents.
Methodological considerations
First, the DiI method used led to labeling of fibers that
branched to form conspicuous terminal fields; this indicates
that the transport times used (7-60 days) were sufficient
for complete labeling of fibers. Labeling was highly reproducible provided that care was taken to ensure that crystals did
not contact neighboring regions. The dye diffused both
anterogradely and retrogradely along processes, as was
shown in reciprocal labeling experiments, and it labeled
axon collaterals regardless of the point of application. This
explains why DiI labeling revealed the complex pattern of
projections of the entopeduncular nucleus after application
into the habenula (see below). This characteristic of the
labeling procedure used, which is similar to that suspected
to occur with wheat germ agglutinin (WGAI-conjugated
horseradish peroxidase (HRP) labeling (Sas et al., 19931,
should be borne in mind when interpreting the present
results. For instance, the terminal fields observed in the
ventral and dorsolateral telencephalon after application of
DiI to the habenula should not be interpreted as habenulotelencephalic projections.
Second, it should be stressed that both axon membranes
and myelin sheaths in the region of dye application were
labeled (in transverse sections appearing as fluorescent
rings). Thus, in cases of habenular application, the posthabenular region contained small, round, labeled cells medial
to the fasciculus retroflexus, in which the presence of
processes was scarcely distinguishable. Likewise, similar
cells were occasionally observed near the medial olfactory
tract, in the region of the IPN, and (less frequently still) in
other regions, always close to tracts with conspicuously
labeled myelinated fibers. Most (if not all) of these round
cells are probably oligodendrocytes that were labeled because their membranes are continuous with myelin sheaths.
The possible labeling of oligodendrocytes was also remarked upon by von Bartheld et al. (1990),and it may lead
to such cells being identified mistakenly as neurons. Interestingly, in areas containing putative oligodendrocytes (close
to the fasciculus retroflexus along its thalamic course and
dorsal to the IPN), a few stained tanycytes were generally
observed. The association of the two types of cell (oligodendrocytes and tanycytes) with tracts containing heavily
labeled myelinated fibers strongly suggests that DiI diffuses
from myelinated sheaths to tanycytes, probably via oligodendrocytes that contact tanycyte processes through gap junctions, as was found recently in a teleost (Diaz-Regueira et
al., 1992). Moreover, the variable number and location of
these cells are further arguments in favor of their nonneuronal character. Regardless of these considerations, this
methodological problem is of limited concern in the present
Holmgren (1920) reported that the habenulae of salmonids are asymmetric, the right habenular nucleus being
larger than the left and the neurons of the left being more
compactly grouped than those of the right; our observations
are in agreement with these findings. Because our DiI
applications affected all of the habenula, distinction between the connections of dorsal and ventral parts was not
possible. Labeling of habenulorecipient nuclei did not reveal differences in innervation between dorsal and ventral
parts; however, we cannot rule out the possibility that
different areas within the habenulae have different connections. The left habenula of coho salmon contains a serotoninimmunoreactive subnucleus (Ekstrom and Ebbesson, 1988);
in the right habenula, on the other hand, serotoninimmunoreactive neurons appear only transiently during
development (Ebbesson et al., 1992). Moreover, only the
ventral portion of the trout habenula contains a significant
number of somatostatinergic fibers (Becerra et al., 1995),
and a heterogeneous distribution of other types of fiber has
also been observed (unpublished results).
Habenular afferents
In trout, most habenular afferents come from specific
nuclei in the telodiencephalic boundary (entopeduncular
nucleus, preoptic nucleus) and the posterior tubercle (tuberculohabenular nucleus). However, afferents from other
regions (supracommissural telencephalon, pineal, parapineal, locus coeruleus) were also observed. In amphibians,
reptiles, and mammals, habenular afferents originate from
several telencephalic, diencephalic, and mesencephalic nuclei (amphibians: Kemali et al., 1980; reptiles: Hoogland,
1982; Diaz and Puelles, 1992b; mammals: Herkenham and
Nauta, 1977; Parent et al., 1981; Van der Kooy and Carter,
1981; Phillipson and Pycock, 1982; Swanson, 1982; Risold
et al., 1994). In what follows, we compare our results with
those reported previously in fish and in other vertebrates.
In particular, we discuss the possible roles of the habenular
circuitry in descending and ascending forebrain pathways.
Our results in trout reveal a major habenulopetal nucleus
rostral to the optic chiasma. This nucleus correspond to the
“area somatica” of Holmgren (1920) and to the entopeduncular nucleus of Sheldon (19121, Bergqvist (19321, Northcutt and Davis (19831, and Gomez-Segade and Anadon
(1988). To our knowledge, this is the first demonstration of
habenular projections of the entopeduncular nucleus in a
teleost. In trout, Vigh-Teichmann et al. (1983) and 01ivereau et al. (1984) identified a somatostatinergic nucleus
located at the level of the anterior commissure and rostral
to it as the entopeduncular nucleus (Becerra et al., 1995);
this nucleus probably corresponds to the nucleus olfactorius lateralis of Sheldon (19121, the nucleus olfactorius
lateralis of Holmgren (19201, or the lateral nucleus of the
area ventralis of Bass (1981a).
Early studies suggested that as many as seven telencephalic tracts project to the habenula in brook trout (tr.
olfactohabenularis, tr. hippocampohabenularis pars lateralis, tr. corticohabenularis, tr. teniae, tr. olfactorius lateralis
habenulae rectus et cruciatus, tr. hippocampohabenularis
pars medialis, and tr. olfactorius medialis habenulae;
Holmgren, 1920). Sheldon (1912) likewise described several
telencephalohabenular tracts and, in addition, a tractus
preopticohabenularis (with partes anterior, medialis, lateralis, and posterior) in the goldfish. No tracts of this type
were detected in the present study. Indeed, our results after
application of dye in the habenula and ventral telencephalon suggest that the “habenulopetal” tracts of Holmgren
(19201, in fact, are entopedunculofugal projections to the
ventral and laterodorsal telencephalic areas.
Experimental studies of olfactory bulb efferents have
indicated that olfactory projections that cross in the habenular commissure are present in some teleost species (Finger,
1975; Bass, 1981b; Northcutt and Davis, 1983; von Bartheld et al., 1984; Levine and Dethier, 1985; Sas et al.,
1993) but not in others (Scalia and Ebbesson, 1971; Davis
et al., 1981; Ebbesson et al., 1981; Murakami et al., 1983;
Prasada Rao and Finger, 1984). Like these latter authors,
we did not find evidence of olfactohabenular fibers in trout.
Even in species in which olfactory fibers use the habenular
commissure, it is not clear whether they make any contacts
in the habenula, although habenular terminal fields have
been reported (Sas et al., 1993).The experiments of Herkenham and Nauta (1977) in the rat indicated that the stria
medullaris contains passage fibers from several olfactory
structures that do not terminate in the habenula. Axons of
some telencephalic lobe neurons in lamprey also cross the
habenular commissure, although they do not seem to
contact habenular neurons (Northcutt and Puzdrowski,
1988; Yaiiez and Anadon, 1994).
Some studies of the bulk telencephalic efferents in a few
teleost species have indicated the existence of habenulopeta1 projections (Striedter, 1990; Villani et al., 1994a).
However, Vanegas and Ebbesson (19801, Echteler and
Saidel (1981), and Murakami et al. (1983) do not mention
telencephalohabenular projections. In trout, we could not
find any evidence of such telencephalohabenular projections: specifically, no labeled cells were found either in the
olfactory bulb or in the precommissural telencephalon.
Moreover, our results suggest that the entopeduncular
nucleus is the only nucleus with a significant habenular
projection, although a small habenulopetal nucleus is also
present in the commissural region. In view of its position,
the entopeduncular nucleus of the trout appears to correspond to the main habenulopetal nucleus of lamprey, the
subhippocampothalamic nucleus (Yafiez and Anadon, 1994);
thus, these nuclei can be considered as homologous.
In the habenula of land vertebrates, most telencephalic
afferents arise from nuclei intercalated in the telencephalic
tracts. In frog, afferents originate ipsilaterally from three
nuclei of the telodiencephalic boundary (the entopeduncular nucleus, the nucleus dorsomedialis anterior thalami,
and the adjacent periventricular area; Kemali et al., 1980).
These nuclei may correspond to the cells of the posterior
pole of the telencephalon and striatal complex found to
project to the habenula in newts (Clairambault et al., 1986).
In lizards, the several telencephalic nuclei projecting to the
medial habenula (nucleus of the posterior pallial commissure, nucleus septalis impar, nucleus eminentia thalami,
and nucleus of the stria medullaris) and to the lateral
habenula (bed nucleus of the anterior commissure, diagonal
band nucleus, and lateral preoptic area) are related to the
main telencephalic tracts (Diaz and Puelles, 1992b). In
mammals, most telencephalic afferents to the habenula
come from nuclei intercalated in the main telencephalic
tracts: the entopeduncular nucleus (Herkenham and Nauta,
1977; Parent et al., 1981; Van der Kooy and Carter, 1981;
Araki et al., 1984), the nucleus of the diagonal band
(Herkenham and Nauta, 1977; Parent et al.,1981; Contestabile and Fonnum, 19831, the inner segment of the pallidum
(Parent and De Bellefeuille, 1982; Hazrati and Parent,
19911, the bed nucleus of the anterior commissure (Staines
et al., 19881, and the nuclei of the posterior (supracommissural) septum (n. septofimbrialis, n. triangularis; Herkenham and Nauta, 1977; Parent et al., 1981; Contestabile and
Fonnum, 1983; Fonnum and Contestabile, 1984; Staines et
al., 1988; Kawaja et al., 1990). The two habenular nuclei of
mammals (the medial and the lateral) can be distinguished
by their different afferents (Herkenham and Nauta, 1977),
and similar results have been obtained in reptiles (Diaz and
Puelles, 1992b). The lack of distinction between medial and
lateral habenular nuclei in trout and the presence of a
single main habenulopetal nucleus in the telencephalon
might be related to the compact organization of the actinopterygian telencephalon, which is strikingly different from
that of other vertebrates (see Northcutt and Braford,
By application of DiI to either the habenula or the pineal
organ, we were able to confirm the existence of pineal
projections to the trout habenula (Hafeez and Zerihun,
1974). Thus, it is conceivable that habenular function is
influenced by light. Pinealohabenular projections have also
been reported on the basis of experimental studies in other
teleosts (Ekstrom, 1984; Ekstrom and van Veen, 1984).
This contrasts with the apparent lack of pineal projections
to the habenula in lampreys (Puzdrowski and Northcutt,
1989; Yadez et al., 1993) and in some amphibians (Paul et
al., 1971; Eldred et al., 1980). Application of DiI to the
habenula or the parapineal organ of trout (Yaiiez et al.,
1996; present results) confirms the existence of parapinealohabenular projections (Rudeberg, 1969).
The posterior tubercle of trout contains a well-developed
habenulopetal nucleus (the tuberculohabenular nucleus).
This nucleus, which is located rostral or somewhat lateral
to the posterior tuberal nucleus itself, extends along the
course of the main telencephalohypothalamic tracts. Thus,
it probably corresponds to the habenulopetal cells located in
the lateral hypothalamic and lateral mammillary areas of
the lizard (Diaz and Puelles, 199213) and, possibly, to the
habenulopetal cells reported from the medial hypothalamus (Parent et al., 1981), the arcuate nucleus (Sim and
Joseph, 1991), and the mammillary region (Veazey et al.,
1982) of mammals. In the lungfish, neurons in the paraventricular organ seem to project to the habenula (von Bartheld and Meyer, 1990); the location of these cells in the
tuberal region suggests that they might correspond to the
trout habenulopetal nucleus, but it should be noted that
they are cerebrospinal fluid contacting. The periventricular
distribution of neurons in the brain of the lungfish, which is
totally unlike the more complex distribution observed in
teleosts, might explain this difference. Habenulopetal cells
have not been detected in this region either in lamprey
(Yafiez and Anadon, 1994) or in amphibians (Kemali et al.,
1980; Clairambault et al., 1986).These results suggest that
a tuberal population of habenulopetal cells might have
appeared independently in teleost, lungfish, and amniote
lineages. However, experimental studies in other vertebrate groups are needed to rule out the possibility that the
lack of a tuberal habenulopetal system in amphibians is a
derived characteristic.
Possible origins of chemically identified
afferent fibers
A number of chemically identified afferent fiber types
have been observed in the salmonid habenula, including
somatostatinergic (Becerra et al., 1995), catecholaminergic
(Manso et al., unpublished observations), serotoninergic
(Ekstrom and Ebbesson, 1988), and FMRF-amide fibers
(Ekstrom et al., 1988; Ostholm et al., 1990). Interestingly,
habenulopetal somatostatinergic (Palkovits et al., 1982),
dopaminergic (Phillipson and Griffith, 1980; Phillipson and
Pycock, 1982; Skagerberg et al., 1984), and serotoninergic
(Steinbusch, 1984) fibers have also been demonstrated in
mammals. Owing to the small number of these fibers
observed in the trout habenula, with the exception of
somatostatin, they probably do not form part of a specific
habenulopetal system, but they may form part of diffuse
projection systems to a number of brain regions.
The size and distribution of DiI-labeled cells in the
preoptic nucleus is similar to that previously reported for
somatostatinergic cells in adult trout (Becerra et al., 19951,
which suggests that the somatostatinergic fibers observed
in the habenula may originate from the preoptic nucleus.
Although the entopeduncular nucleus gives rise to habenulopetal somatostatinergic projections in mammals (Vincent
and Brown, 1986), the trout entopeduncular nucleus does
not contain somatostatin-immunoreactive cells (Becerra et
al., 1995),and it is currently unclear whether the entopeduncular nuclei of mammals and teleosts are homologous.
For some of the other fiber types, our DiI applications
likewise indicate a possible nucleus of origin. The cells
labeled in the locus coeruleus are very similar in location
and appearance to the large tyrosine hydroxylase-immunoreactive neurons present in this region of the trout brain
(Manso et al., 1993). In teleosts, locus coeruleus cells form a
diffuse noradrenergic projection system to most brain
regions (Ekstrom et al., 1986; Hornby and Piekut, 1990;
Meek et al., 1993; Ma, 1994).
Serotoninergic fibers were scarce in the trout habenula.
Six areas containing serotoninergic cell bodies have been
demonstrated in the diencephalon and mesencephalon of
rainbow trout (Frankenhuys-van den Heuvel and Nieuwenhuys, 19841, and the pineal organ also contains serotoninergic cells (van Veen et al., 1984).Although the posthabenular region, the nucleus anterior tuberis, the periventricular
region of the tuberal periventricular region, and the raphe
nuclei of teleosts contain a rich population of serotoninergic
cells (Frankenhuys-van den Heuvel and Nieuwenhuys,
1984; Meek and Joosten, 1989; Johnston et al., 19901, we
did not observe DiI-labeled neurons in any of these locations. The present demonstration of pinealohabenular projections strongly suggests that the serotoninergic fibers in
the rainbow trout habenula arise from the pineal organ.
Although serotoninergic cells have been observed in the left
habenula of coho salmon (Ekstrom and Ebbesson, 19881,
such cells were not detected in rainbow trout (Frankenhuysvan den Heuvel and Nieuwenhuys, 1984).
Scarce FMRF-amide-immunoreactive fibers were observed in the trout habenula (Ekstrom et al., 1988); however, it is not clear whether these terminate in, or simply
pass through, the habenula. There are a number of FMRFamide-immunoreactive nuclei in the telencephalon, diencephalon, and mesencephalon of teleosts (Fujii and Kobayashi, 1992; Vecino and Ekstrom, 1992);in the present study,
however, application of DiI to the habenula did not lead to
labeling of neurons in any of these previously reported
locations. In salmon, gonadotropinreleasinghormone (GnRHIimmunoreactivefibers are also present in the habenula (Amano
et al., 1991), although, again, it is not clear whether they
originate in terminal ganghon or in telencephalic or preoptic
GnRH-immunoreactiveneurons. The fact that application of
DiI to the trout habenula did not lead to labeling of known
FMRF-amide- or GnRH-immunoreactive nuclei may simply
reflect the scarcity of this type of fiber in the habenula.
Habenular efferents
Similar to mammals (Herkenham and Nauta, 19791,
frogs (Kemali et al., 1980), and lamprey (Yafiez and Anadon, 1994),the habenular efferents of trout run in the FR.
This is in good agreement with the results of immunocytochemistry and habenular ablation in goldfish (Villani et al.,
1987, 1991, 1994a,b). The trout FR seems to project not
only to the IPN (Villani et al., 1987, 1991, 1994a,b) but also
to the isthmal raphe. A similar distribution of the FR, with
fibers extending caudally into the isthmal raphe, has been
reported in lampreys (Yanez and Anadon, 1994), amphibians (Kemali and Lazar, 1985), lizards (Distel and Ebbesson, 1981; Diaz and Puelles, 1992a),and mammals (Herkenham and Nauta, 1979). Interestingly, the habenular
projections to the interpeduncular and raphe nuclei appear
to correspond only to the core of the FR of reptiles and
mammals, which originates from the medial habenular
nucleus (Distel and Ebbesson, 1981; Diaz and Puelles,
1992a; Herkenham and Nauta, 1979; Contestabile and
Flumerfelt, 1981; Hamill and Jacobowitz, 1984; Contestabile et al., 1987).
In lizards and mammals, the FR also projects fibers to
mesencephalic, diencephalic, and telencephalic nuclei (mammals: Herkenham and Nauta, 1979; lizard: Distel and
Ebbesson, 1981; Diaz and Puelles, 1992a). Interestingly,
projections to the periaqueductal gray (Beat et al., 19901,
substantia nigra (Christoph et al., 19861, and ventral
tegmental area (Christoph et al., 1986; Oades and Halliday,
1987) arise from the lateral habenular nucleus and run in
the mantle of the FR; similar observations have been made
in lizards (Distel and Ebbesson, 1981). Together, these
results suggest that there is no nucleus homologous to the
lateral habenular nucleus either in trout (present results)
or in lamprey (Yafiez and Anadon, 1994).Interestingly, the
lateral habenular nucleus of mammals receives afferents
from telencephalic nuclei that do not project to the medial
habenula, like the entopeduncular nucleus (Herkenham
and Nauta, 1977; Van der Kooy and Carter, 1981; Araki et
al., 1984; Vincent and Brown, 1986) and the globus pallidus
(Parent and De Bellefeuille, 1982; Hazrati and Parent,
1991). Again, similar observations have been made in
lizards (Hoogland, 1982; Diaz and Puelles, 1992b). Thus,
the lateral habenular nuclei of lizards and mammals might
be a relatively recent development in vertebrate phylogeny,
perhaps reflecting the increasing importance of cortical
circuits. If so, it seems likely that the land vertebrate
homologue of the principal habenulopetal nucleus in trout
(the entopeduncular nucleus) is not the entopeduncular
nucleusiglobus pallidus, but, rather, it may be the septa1
nuclei, which, in lizards and in mammals, project to the
medial habenula.
A further problem when analyzing the habenular projections of trout after DiI labeling is the interpretation of
fibers observed in some thalamic, pretectal, and tegmental
regions. In experiments in which DiI was applied to the
pineal vesicle, all of these areas received a rich innervation,
in agreement with results of Hafeez and Zerihun (1974).
The pattern of fibers in these regions is similar regardless of
whether DiI is applied to the habenula or to the pineal
vesicle, which suggests that the fibers in thalamic, pretectal, and tegmental regions found when DiI is applied to the
habenula are pineal fibers that become labeled through
their habenulopetal collaterals.
In conclusion, our observations suggest that the habenular system of fishes forms a system of projections between
nuclei of the caudal telencephalon and posterior tubercle
and the basal rnesencephalonirostral medulla. This system
is simpler than that found in amniotes. Comparison of
teleosts with amniote species in which the habenular
system has been studied experimentally indicates that their
pattern of connections is similar only to the medial habenular nucleus of reptiles and mammals. These land vertebrates appear to have acquired additional habenular circuitry with the appearance of the lateral habenular nucleus.
Further study of habenular connections in other groups of
fishes may shed light on whether the absence of a lateral
habenular nucleus homologue in teleosts is a primitive or a
derivative characteristic.
The authors thank Mr. G. Norman for his stylistic
suggestions for improving the paper and Miss C. Farifia for
providing the trouts. This work was supported by grants
from the DGICYT (93-0527CO201) and the Xunta de
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