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Afferent and efferent connections of the parapineal organ in lampreys A tract tracing and immunocytochemical study

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THE JOURNAL OF COMPARATIVE NEUROLOGY 403:171–189 (1999)
Afferent and Efferent Connections of the
Parapineal Organ in Lampreys: A Tract
Tracing and Immunocytochemical Study
JULIÁN YÁÑEZ,1 MANUEL ANGEL POMBAL,2 AND RAMÓN ANADÓN3*
1Department of Cell and Molecular Biology, Faculty of Sciences,
University of La Coruña, 15007-La Coruña, Spain
2Department of Functional Biology and Health Sciences, Faculty of Sciences,
University of Vigo, 36200-Vigo, Spain
3Department of Fundamental Biology, Faculty of Biology, University of Santiago de
Compostela, 15706-Santiago de Compostela, Spain
ABSTRACT
The neural connections of the parapineal organ of two species of lampreys were studied
with the fluorescent dye 1,18-dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate
(DiI) and with immunocytochemistry. The lamprey parapineal organ consists of a vesicle and
a ganglion that are connected to the left habenula. Labeling experiments included the
application of DiI to the parapineal organ, left and right fasciculus retroflexus, left habenula,
and the left pretectal region. Afferent parapineal fibers run in the left fasciculus retroflexus to
the interpeduncular nucleus. The parapineal fibers of this fascicle arose from parapineal
ganglion cells, whereas DiI application to the left habenula labeled both neurons of this
ganglion and bipolar cells in the parapineal vesicle. Efferent neurons were observed in the left
habenula, and bilaterally in the subhippocampal nucleus and the dorsal pretectum. Labeling
with DiI also revealed a hippocampal projection.
Immunocytochemical study of the parapineal vesicle revealed serotonin-immunoreactive
cells in both species of lamprey, as well as substance P–immunoreactive (SP-ir) cells in sea
lamprey and choline acetyltransferase–immunoreactive (ChAT-ir) cells in the river lamprey.
The SP-ir cells and ChAT-ir cells formed a rich neuropil in the parapineal ganglion. Calretinin-ir
cells were numerous in the ganglion. Neuropeptide Y–immunoreactive and ␥-aminobutyric
acid–immunoreactive efferent fibers were observed in the parapineal organ. Neuropeptide
Y–immunoreactive fibers originate in the subhippocampal nucleus, whereas ␥-aminobutyric
acid–immunoreactive fibers might also arise in the pretectal nucleus. A few galanin-ir fibers
were observed. These results indicate that the parapineal connections are completely different
from those of the pineal organ. The possible homology between parapineal organs of
vertebrates is discussed. J. Comp. Neurol. 403:171–189, 1999. r 1999 Wiley-Liss, Inc.
Indexing terms: pineal complex; DiI; habenula; entopeduncular nucleus; GABA; Agnathans
The pineal complex of lampreys consists of two photoreceptive organs, the pineal and parapineal organs (Meiniel,
1969; Meiniel and Collin, 1971; Cole and Youson, 1982).
The pineal organ consists of a vesicle joined to the posterior commissure by a long pineal stalk, whereas the
parapineal organ consists of a parapineal vesicle and a
ganglionic mass (parapineal ganglion) associated with the
tela choroidea, which extends between the parapineal and
the habenula and through which runs a diffuse parapineal
tract. It is notable that no similar ganglion has been found
in other vertebrates. The nomenclature given to the ganglion and the tract entering the left habenula reflects two
different interpretations. According to Studnicka (1905),
r 1999 WILEY-LISS, INC.
the parapineal ganglion should be considered as a part of
the left habenula and, therefore, should be called the
accessory habenular nucleus, and the tract, the habenular
Grant sponsor: Spanish Education Ministry; Grant number: PB96–0945C03; Grant sponsor: Xunta de Galicia; Grant number: XUGA20012B96;
Grant number: XUGA20002B97; Grant sponsor: University of Vigo; Grant
number: 64102C734.
*Correspondence to: Dr. R. Anadón, Department of Fundamental Biology,
Faculty of Biology, University of Santiago de Compostela, Santiago de
Compostela 15706, Spain. E-mail: bfanadon@usc.es
Received 27 January 1998; Revised 5 August 1998; Accepted 17 August
1998
172
J. YÁÑEZ ET AL.
tract. Meiniel and Collin (1971) support this interpretation
on the basis of the similar ultrastructural appearance of
the cells of the ganglion and of the left habenula. According
to Tretjakoff (1915), however, the parapineal ganglion and
parapineal tract are different structures from the habenula. This question could be clarified by studying the
connections of the parapineal organ. An experimental
tracing study of the lamprey pineal complex has indicated
that it has projections to the pretectum and mesencephalic
tegmentum, without any distinction of pineal and parapineal components (Puzdrowski and Northcutt, 1989). Another study in larval lamprey (Yáñez et al., 1993) reported
similar projections for the pineal organ.
With regard to its function, the pineal complex, as a
whole, has been considered as a sensory organ that
conveys photic information to neural structures (Morita
and Dodt, 1973, Pu and Dowling, 1981, Tamotsu and
Morita, 1986; Samejima et al., 1989) and releases hormonal messages (Joss, 1977; Bolliet et al., 1993) that may
be involved in sexual maturation (Joss, 1973a), changes in
body coloration (Joss, 1973b, 1977), circadian rhythms
(Morita et al., 1992), and metamorphosis (Cole and Youson, 1981). Immunocytochemical studies of the lamprey
parapineal organ have reported the expression of molecules involved in light signal transduction (Vigh-Teichmann et al., 1983; Kuo et al., 1988; Tamotsu et al., 1990,
1994; Garcı́a-Fernández et al., 1997) and the presence of
serotonin (a possible precursor of melatonin: Meiniel,
1978, 1980; Tamotsu et al., 1990), somatostatin (Yáñez et
al., 1992), and glutamate (Debreceni et al., 1997), but no
particular function has been assigned to this structure
that is generally considered as a rudimentary or regressed
sense organ (Cole and Youson, 1982).
The aim of the present investigation with carbocyanine
dye tracing was to determine the relation between the
parapineal ganglion and vesicle with the habenula and
other parts of the brain. In addition, the immunocytochemical study of the neuropeptides, neurotransmitter, and
neurotransmitter synthesizing enzymes aimed to investigate the chemical nature and possible origins of some fiber
systems of the parapineal organ. Because lampreys are
living representatives of the earliest vertebrates, the Agnathans, better knowledge of the parapineal organ is
Abbreviations
FR
H
HY
IPN
LH
MO
OB
OC
ON
OT
P
PH
PP
PC
PT
RH
SH
ST
TC
TH
TL
TO
fasciculus retroflexus
habenula
hypothalamus
interpeduncular nucleus
left habenula
medulla oblongata
olfactory bulb
optic chiasma
optic nerve
optic tract
pineal organ
primordium hippocampi
parapineal organ
posterior commissure
parapinealopetal pretectal nucleus
right habenula
subhippocampal-thalamic nucleus
striatum
tela choroidea
thalamus
lateral telencephalic lobe
optic tectum
likely to contribute to understanding early evolution of the
pineal complex.
MATERIAL AND METHODS
Animals
Adult river lamprey (Lampetra fluviatilis) were caught
in the spring in Söderham (Sweden) during their upstream
migratory phase. They were maintained at 4–10°C in
well-aerated freshwater aquaria before experiments. Upstream migrating adults, young adults, and ammocoetes of
sea lamprey (Petromyzon marinus) were caught in the
Rivers Miño and Ulla (northwest Spain) and maintained
at room temperature. Before fixation, all lampreys were
deeply anesthetized with tricaine methane sulfonate
(Sigma, St. Louis, MO) and killed by decapitation. All the
experiments conformed to the European Community’s
guidelines on animal care and experimentation. The brains
were then quickly exposed to fixative by carefully removing the skull roof to preserve the pineal complex.
DiI labeling
Twelve adult lampreys (L. fluviatilis, L.) and 6 young
postmetamorphic sea lampreys (Petromyzon marinus, L.)
were used for DiI labeling. The brain was fixed in cold 4%
paraformaldehyde in 0.1 M phosphate buffer (PB), pH
7.2–7.4. Crystals of 1,18-dioctadecyl-3,3,38,38-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, Eugene, OR) were applied either to the parapineal organ
(three river lampreys, one sea lamprey), the left habenula
(three river lampreys, one sea lamprey), the pretectal
region (two river lampreys), the right fasciculus retroflexus (one river lamprey, three sea lampreys), or the left
fasciculus retroflexus (three river lampreys, one sea lamprey) following the method of Yáñez et al. (1996). In brief,
after fixation, the brain was embedded in 3% agarose, and
the block was cut on a Vibratome to the desired level. A
small crystal of DiI was then applied with the tip of an
electrolytically sharpened insect pin or a micropipette.
Then, the entire surface was sealed with melted agarose to
avoid spillage of crystals, and the brain was left in fresh
fixative for 7–20 days at 37°C in darkness. Vibratome
sections (50- to 100-µm thick) of the brain were examined
and photographed with a fluorescence microscope equipped
with a rhodamine filter set.
To reveal the parapineal connections, the parapineal
organ was labeled following the previously described procedure. In an additional experiment, the tracer was applied
to the parapineal vesicle without embedding the brain in
agarose or cutting any part of the brain. To confirm the
results of these experiments, DiI was applied to those
projecting areas visualized: the caudal left habenula, the
left and right fasciculus retroflexus at the level of the
infundibulum, and the left pretectum at a level caudal to
the posterior commissure. The nomenclature used in this
study to name the brain areas follows that of Heier (1948)
and Schöber (1964).
Immunocytochemistry
For immunocytochemistry, the brains were rapidly dissected out and fixed by immersion in the fixative appropriate for each antibody used (see also Pombal et al., 1997). A
solution of 3% glutaraldehyde and 0.5% paraformaldehyde
in PB or 5% glutaraldehyde in PB were used for ␥-aminobu-
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
tyric acid (GABA; eight river lampreys, two sea lampreys);
4% paraformaldehyde in PB for choline acetyltransferase
(ChAT; four river lampreys); 4% paraformaldehyde and 0.2
picric acid in PB for serotonin (5-HT; five river lampreys,
two sea lampreys), and substance P (SP; two sea lampreys,
two river lampreys) and Bouin’s fluid for calretinin (CR;
three river lampreys, two sea lampreys), neuropeptide Y
(NPY; three river lampreys), and galanin (six river lampreys). Those brains fixed in Bouin’s fluid were subsequently dehydrated, embedded in paraffin, and cut (15-µmthick) either in the transverse, horizontal, or sagittal
plane. The other brains were cryoprotected with 30%
sucrose, frozen with liquid nitrogen, sectioned (15-µmthick) in the same planes on a cryostat, and processed
according to the immunoperoxidase technique. Briefly,
sections were incubated with one of the primary antisera
raised in rabbits against GABA (Chemicon, Temecula, CA;
Affinity, Mamhead, U.K.; or Sigma; Pombal et al., 1997;
dilution, 1:1,000 to 1:2,000), 5-HT (Zhang et al., 1996;
dilution 1:1,000), SP (Pombal et al., 1997; dilution 1:2,000),
CR (7696, Swant, Bellinzona, Switzerland; Schwaller et
al., 1993; dilution, 1:1,000), NPY (Sigma; dilution, 1:600),
or galanin (Jiménez et al., 1996; dilution 1:1,500). They
were subsequently incubated with goat anti-rabbit (Dakopatts, Glostrup, Denmark; dilution, 1:60) and rabbit
peroxidase-antiperoxidase complex (Dakopatts; dilution,
1:300). The antisera were diluted in 0.1 M phosphatebuffered saline, pH 7.2–7.4, containing 0.3% Triton X-100.
The peroxidase-antiperoxidase complex was developed by
incubation in 3,38-diaminobenzidine (Sigma; 50 mg/100
ml) and 0.005% hydrogen peroxide in 0.05 M Tris buffer,
pH 7.3. The same buffer was used to stop the reaction. For
ChAT immunocytochemistry, the procedures used are the
same as in Marı́n et al. (1997), by using a goat anti-ChAT
serum (Chemicon; dilution, 1:100).
RESULTS
The distribution of afferents and efferents of the parapineal organ of lampreys revealed after DiI application to
this structure or to some of its targets will be described
first. A detailed description of the structures of the parapineal system immunoreactive for some neurotransmitters,
neurotransmitter synthesizing enzymes, neuropeptides,
and a calcium binding protein, are described next.
Tract tracing methods
The application of the carboindocyanine dye DiI to fixed
tissue allows the tracing of connections of minute structures without affecting neighboring regions. As shown in
Figure 1, the procedure of DiI application to the parapineal
organ of lamprey specifically labeled this organ, and did
not affect the pineal organ or any other brain structure. In
two of the experiments the intention was to label only the
parapineal vesicle, but this also led to labeling of the
ganglion. Thus, the connections revealed by these experiments refer to the entire parapineal organ.
Afferent connections
Application of DiI to the parapineal organ. DiI application to the parapineal organ labeled the parapineal tract in
the tela choroidea, the left (small) habenula (both cells and
fibers), the left fasciculus retroflexus, and the efferent
fibers running through the right habenula and the thalamus. The cells and neuropil of the right (large) habenula
and the right fasciculus retroflexus were unlabeled. Both
173
cells and fibers were labeled in the left habenula, suggesting that the parapineal organ and left habenula are
bidirectionally connected (see below). In addition, two
efferent neuronal groups (located in the caudal telencephalon and the pretectum, respectively) were labeled bilaterally, as was a conspicuous tract that runs to the primordium hippocampi. The application of DiI to the parapineal
organ did not produce any labeling in the pineal organ. The
targets of the parapineal and the efferent nuclei, with the
exception of the pretectal nucleus, thus, coincide with
those reported for the habenula in larval sea lamprey
(Yáñez and Anadón, 1994). Note that the connection between the parapineal and the habenula was not observed
in that study.
The parapineal tract consisted of numerous small nerve
fascicles running parallel to each other in the tela choroidea (Fig. 1B). The left habenula was strongly fluorescent
because of the presence of numerous labeled fibers, but a
few labeled neurons were also observed (Fig. 1C,D). The
labeling of this habenula contrasted with the lack of
labeling of cells and fibers in the right habenula (excluding
the efferent fibers that cross it caudally) (Fig. 1C). The left
fasciculus retroflexus was intensely labeled. The fibers of
the tract run compactly, and very few labeled fibers diverge
from the fascicle, with the exception of a few fibers running
in the dorsal thalamus-pretectum, caudal to the habenula.
In the interpeduncular nucleus the fibers of this fascicle
formed a conspicuous unpaired terminal field, probably
branching and crossing the midline several times (Fig.
2A-E). In the midline, some fibers exhibit large irregular
expansions (Fig. 2D). The parapinealofugal fibers extended only to the rostral part of the interpeduncular
nucleus, and no fibers were seen to extend as far as the
rhombencephalon proper.
Application of DiI to the left habenula or the left fasciculus retroflexus. To determine whether the afferent fibers
labeled after DiI application to the parapineal originate
from the parapineal vesicle, the parapineal ganglion, or
both, three types of labeling were carried out: one in the
left habenula and the other two in the left or right
fasciculus retroflexus at the level of the posterior tubercle.
Application of DiI to the right fasciculus retroflexus produced no labeling of parapineal structures. Application of
DiI to the left habenula or the left fasciculus retroflexus led
to intense labeling of the parapineal ganglion, indicating
that cells of this ganglion give rise to the afferent fibers
running in the left fasciculus retroflexus (Fig. 3A-G). The
application of DiI to the left habenula also led to intense
labeling of numerous cells in the parapineal vesicle
(Fig. 3A-D), whereas labeled cells were not observed in the
vesicle after application to the left fasciculus retroflexus.
The labeled cells in the parapineal vesicle were spindleshaped, with a dendrite extending to the parapineal lumen
and ending in a small head, sometimes bearing an irregular apical process, and a basal process. Careful observation
of the basal process suggests that it may branch at the
transition to the parapineal ganglion, indicating that the
labeled bipolar (long axon) cells might project both to the
parapineal ganglion and left habenula. No fiber or cell of
the pineal organ became labeled in these three sets of
experiments, in agreement with the previously reported
findings for the lamprey pineal organ (Yáñez et al., 1993).
Efferent connections. Application of DiI to the parapineal organ led to labeling of fibers that run through the
thalamus lateral to both habenulae and through the right
174
Fig. 1. DiI labeling of the parapineal organ of river lamprey.
A: Transverse section showing the labeled parapineal vesicle (black
asterisk) and parapineal ganglion (black star) just caudal to the
application point. Note that both the pineal organ and dorsal telencephalon (white star) are unlabeled. B: Flat view of the tela choroidea
between the parapineal ganglion and the habenula showing the
fascicles of the parapineal tract. The position of the unlabeled pineal
stalk, which runs in a groove of the tela, is also distinguishable
J. YÁÑEZ ET AL.
(arrow). C: Strong labeling of the left habenula (wide arrow) and of
efferent fibers running through the thalamus and crossing the right
habenula (long arrows). The small arrows indicate labeled neurons;
dashed lines indicate the borders of the brain. D: Detail of the left
habenula showing strong labeling of neuropil and a few habenular
neurons (arrows). For abbreviations, see list. Scale bars ⫽ 50 µm in
A,B,D, 100 µm in C.
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
175
Fig. 2. Transverse sections of the interpeduncular nucleus of river
lamprey showing labeled parapineal afferents. A: Transition from the
left fasciculus retroflexus (arrow) to the rostral part of the interpeduncular nucleus. B: Rich plexus of parapinealofugal fibers in the rostral
interpeduncular nucleus. C: Detail of the branching of parapineal
fibers (arrow). D: Lateral expansions of parapineal fibers in the
midline (arrow). E: Transition to the caudal part of the interpeduncular nucleus showing the end of parapineal fibers. Note the ventral
unlabeled portion of the nucleus (asterisk). Star, raphe. Scale bars ⫽
100 µm A,B,E; 50 µm in C,D.
habenula in the habenular commissure (Fig. 1C). In the
rostral thalamus, they formed a tract that apparently
bifurcated between the primordium hippocampi and the
subhippocampal nucleus, the rostrodorsal branch extending, as a band-shaped tract, through the inner part of the
primordium hippocampi and ending freely, whereas the
ventral branch was followed to a sparse population of
small labeled neurons of the lobus subhippocampalisrostral thalamus which appears to give rise to these fibers
(Fig. 4A–D). These cells have long thin dendrites that run
through the neuropil. A few labeled cells of this population
may even reach the ventral limit of the primordium
hippocampi. This population corresponds to the subhippocampal-thalamic habenulopetal nucleus of Yáñez and
Anadón (1994).
Application of DiI to the parapineal organ also led to
labeling of a nucleus of small neurons in the middle of the
pretectal region, which extends rostrocaudally near the
dorsal limit of the optic tract (Fig. 5A,B). The labeled cells
of this nucleus exhibit long thin dendrites extending
dorsolaterally to the subpial surface and forming a conspicuous dendritic plexus. Application of DiI to the region
of the pretectal nucleus produced labeling of fibers in the
habenulae and the proximal parapineal tract (Fig. 5C,D).
This suggests that the pretectal nucleus is also habenulopetal. Unfortunately, in the experiments of this type, the
tela choroidea was broken and fibers from the pretectal
region could not be followed to the parapineal organ. A
schematic representation of the afferent and efferent connections of the lamprey parapineal is shown in Figure 6.
Immunocytochemistry of the
parapineal organ
To characterize the chemical nature of parapineal circuits, we carried out an immunocytochemical study with
antisera to several neurotransmitters, neurotransmittersynthesizing enzymes, and neuropeptides, as well as to the
176
J. YÁÑEZ ET AL.
Figure 3
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
calcium-binding protein calretinin. Most of the results
presented here were for the river lamprey, but some
results were obtained by using sea lamprey. In the parapineal organ, we found immunoreactivity with antisera to
the following substances: 5-HT, SP, ChAT, NPY, GABA,
galanin, and CR.
Cells. In the sea lamprey, but not in the river lamprey,
the walls of the parapineal vesicle contained SP-immunoreactive (SP-ir) bipolar cells. The SP-ir cells, in general,
stained faintly, and were more numerous in the dorsal,
rostral, and lateral regions of the vesicle (Fig. 7A,B). These
pale cells gave rise to more intensely stained fibers running from the outer region of the wall to the medial
neuropil of the ganglion where they form a very rich SP-ir
plexus. Only a few SP-ir fibers were observed in other
neuropil areas of the ganglion and in the tela choroidea
between the parapineal and the habenula. In the river
lamprey, a few SP-ir fibers (probably efferent fibers) were
observed in the ganglion and the tela choroidea, whereas
the vesicle lacks any SP-immunoreactive structure.
ChAT immunoreactivity has been observed in two groups
of bipolar cells of the parapineal vesicle of river lamprey
(Fig. 7C,D). One of the groups, the most numerous, occupies the dorsorostral wall of the vesicle, the other the
ventrocaudal region. Fibers originated from these groups
form numerous ChAT-immunoreactive (ChAT-ir) boutons
among cells of the vesicle and the ganglion. No ChAT-ir
fibers appear to run in the parapineal tract.
5-HT-immunoreactive (5-HT-ir) cells were mostly located in the dorsal, rostral, and lateral regions of the
vesicle and were practically absent from the ventral wall
(Fig. 8A,B). They were bipolar cells with a short process
directed to the lumen and a basal process running near the
pial surface. A few 5-HT-ir fibers enter the parapineal
ganglion. No 5-HT-ir fiber was observed leading from the
parapineal organ to the habenula. 5-HT immunoreactivity
was observed in cells of the parapineal vesicle of both
species of lamprey, but they were very scarce in the sea
lamprey.
CR-immunoreactive (CR-ir) neurons were observed in
the parapineal ganglion of river lamprey but not in the
vesicle (Fig. 8C). The CR-ir cells occupied rostral and
lateral regions of the ganglion, where they may represent
most of the cells. However, in caudal regions, there are
many immunonegative cells. The positivity to CR was
Fig. 3. Transverse sections showing the labeling of the parapineal
organ after application of DiI to the left habenula (A–D) and the left
fasciculus retroflexus (E–G). A: Labeling of bipolar cells ventral in the
parapineal vesicle and strong labeling of the ganglion (black star) of a
young sea lamprey. B: Differential interference contrast micrograph of
the same section showing the pineal and the parapineal (asterisk)
vesicle and ganglion (black star). C: Labeled bipolar neurons (arrow)
in the parapineal vesicle of a river lamprey. Note also the strong
labeling of the ganglion (star). D: Detail of a bipolar cell in the
parapineal vesicle of sea lamprey showing the apical process (arrow)
and possible branching (arrowhead) of the basal process. Star, parapineal ganglion. E: Transverse section through the posterior tubercle
showing the point of DiI application to the left fasciculus retroflexus
(black arrow). Star, optic tract; thin white arrow, posthabenular
region; wide white arrow, posterior hypothalamic recess. F–G: Two
different focal planes of a section of the parapineal ganglion (stars) in
the same experiment as in E, showing the dendritic plexus, with some
dendrites even extending toward the vesicle (asterisk), and labeled
perikarya (small arrows in G). P, pineal organ. Scale bars ⫽ 50 µm in
A-D,F,G; 250 µm in E.
177
weak but clear in perikarya, and rather diffuse in the
neuropil and the parapineal tract. The left habenula
exhibits large areas of CR-ir cells with a similar appearance (Fig. 8D), whereas immunoreactivity to CR in the
right habenula is observed as patches of darker-stained
cells.
Efferent fibers. GABA-ir efferent fibers were observed
in the parapineal organ of both species of lamprey, and
NPY-immunoreactive (NPY-ir) efferent fibers were also
observed in the river lamprey. These fibers enter the
parapineal ganglion, as part of the parapineal tract and
form a rather rich plexus of beaded fibers among its cells
and in the neuropil (Figs. 9A-C, 10A,B). GABA-ir fibers,
which appear to be the most numerous, also enter the
walls of the vesicle in small number (Fig. 9A,B). Although
double labeling was not carried out, comparison of the
distribution of immunoreactive neurons in series of lamprey brains immunocytochemically stained for GABA or
NPY with the retrogradely labeled nuclei after DiI application to the parapineal organ, allowed determination of the
probable origin of either GABA- or NPY-ir efferent parapineal innervation. Many cells of the parapinealopetal subhippocampal-thalamic nucleus exhibited NPY immunoreactivity (Fig. 10C), indicating that this nucleus is the most
probable origin of NPY-ir efferent fibers of the parapineal
ganglion. Small GABA-ir neurons were observed both in
the subhippocampal-thalamic nucleus and the region of
the pretectal parapinealopetal nucleus (not shown), suggesting that efferent GABA-ir fibers of the parapineal
organ may have a dual origin.
Very few (one to four) galanin-ir fibers were observed to
run through the parapineal tract and the ganglion of river
lamprey, but not the vesicle (Fig. 10D,E). The possible
origin of these galanin-ir fibers was not determined because none of the galanin-ir brain nuclei reported in a
previous study of the river lamprey (Jiménez et al., 1996)
corresponds with the efferent nuclei demonstrated by DiI
application to the parapineal organ.
DISCUSSION
Technical comments
DiI labeling is a very sensitive method for tracing
neuronal projections (Holmqvist et al., 1992). When using
this technique in unmyelinated systems, clear and highly
reproducible results can be obtained with a small number
of animals (Yáñez and Anadón, 1996; Yáñez et al., 1993,
1996, 1997). Because this tracer diffuses along membranes
of cell processes both anterogradely and retrogradely from
the application site and bifurcation points, additional
experiments may be necessary to confirm the afferent or
efferent character of some labeled fiber systems. Moreover,
because of the small size and closeness of the two parts of
the parapineal organ (vesicle and ganglion), DiI application to these structures could not clearly differentiate their
respective connections. Therefore, additional labeling of
some parapineal targets, i.e., left habenula and left fasciculus retroflexus, was required to distinguish their respective connections. The results of GABA and NPY immunocytochemistry also served as a control of the results with DiI
tracing on efferent systems, and excluded the possibility
that these systems were labeled by transneuronal diffusion of DiI. From our previous experience with the DiI
method (Yáñez et al., 1993, 1996, 1997; Yáñez and Anadón,
1994, 1996), it does not appear that transneuronal diffu-
178
Fig. 4. Transverse sections through the telencephalon showing
neurons and fibers labeled after DiI application to the parapineal
organ. A: Labeled neurons in the subhippocampal nucleus (SH). The
arrow indicates the subhippocampal sulcus. B: Detail of labeled cells
of this nucleus and their processes. C: Section at the region where
parapinealopetal fibers bifurcate, giving rise to a collateral hippocam-
J. YÁÑEZ ET AL.
pal bundle (wide arrow). The thin arrow indicates the subhippocampal
sulcus. D: Bundle of collaterals of parapineal efferent fibers running
rostralward in the primordium hippocampi (PH). Asterisk, telencephalic ventricle; star, subventricular layer of neurons. In all figures,
medial is to the left. Scale bars ⫽ 100 µm in A,C,D, 50 µm in B.
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
179
Fig. 5. Transverse sections showing parapinealopetal neurons of
the pretectum (PT) labeled after DiI application to the parapineal
organ (A,B), and habenulopetal and parapinealopetal fibers after
application of DiI to the pretectum (C,D). A,B: Rostral and caudal
sections through the pretectum showing the parapinealopetal cells
(thin arrows) and their dendritic plexuses. In A, note the labeled left
fasciculus retroflexus (wide arrow). Asterisk, mesencephalic ventricle.
C: Fibers labeled in the left (asterisk) and right habenulae (RH). Thick
arrow, fiber passing to the parapineal tract; thin arrow, bundle of
labeled fibers entering the right habenula. D: Labeled fibers (arrow) in
the parapineal tract. The tela choroidea (asterisk) was obliquely
sectioned; black star, dorsal telencephalon. Scale bars ⫽ 100 µm in
A,B, 50 µm in C,D.
sion was a relevant problem, providing that the fixation
was good.
those reported for the entire pineal complex in silver
lamprey (Puzdrowski and Northcutt, 1989). Although these
authors assumed that they labeled both the pineal and
parapineal organs, in the light of the present results and
those of Yáñez et al. (1993) on the projections of the
lamprey pineal organ, it is evident that the projections
reported by them correspond only to those of the pineal
organ. The parapineal organ is connected to the habenulo-
Connections of the lamprey parapineal
organ and the question of homology
The pineal and parapineal organs appear to have very
different connections in lamprey. The neural projections of
the parapineal organ reported here differ completely from
Fig. 6. Schematic drawings of a lateral and a dorsal view of the
brain of the lamprey (A,B) and four transverse sections through the
brain (C–F) to show the location of the pineal complex and the
distribution of parapineal-related structures (the pineal complex is
not represented in B). The shaded areas in A and B represent the
terminal fields of parapinealofugal fibers, other fibers were only
represented in C–F. Single arrows indicate the pineal tract, double
arrows represent the parapineal tract, and dots represent the location
of parapinealopetal neurons. The vertical bars (C–F) in A indicate the
levels of the sections. In A,B, rostral is at the left; in C–F, the left side of
the brain is at the right. For abbreviations, see list.
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
181
Fig. 7. Parapineal organs of sea lamprey (A,B) and river lamprey
(C,D) immunostained for substance P (SP) and choline acetyltransferase (ChAT), respectively. A,B: Transverse sections through the rostral
region of the vesicle and ganglion (A), and through the junction
between the vesicle and ganglion (B). Note the pale SP-immunoreactive (SP-ir) cells (arrowheads in A and B), the abundance of SP-ir fibers
in the medial neuropil of the ganglion (arrows in B) and their scarcity
in the rostral neuropil (A). C: Transverse section through the vesicle
showing the rostral group of ChAT-ir cells in the dorsal wall. D: Sagittal
section through the parapineal organ showing the rostrodorsal (outlined arrow) and caudoventral (thin arrow) groups of ChAT-ir cells
(rostral is at the left). Note the numerous boutons in both the vesicle
and ganglion. Asterisks, parapineal vesicle; stars, parapineal ganglion. Scale bars ⫽ 50 µm in A–D.
interpeduncular system, whereas in lampreys the pineal
organ has no habenular connection and instead has projections to other diencephalic and mesencephalic regions. The
origin of the efferents to the pineal and parapineal organs
(a few tegmental neurons for the pineal, and the subhippo-
campal-thalamic nucleus and the pretectal nucleus for the
parapineal) is also completely separate. Therefore, these
two organs do not seem to be directly related in lampreys
and are probably involved in modulation of different
systems by light.
182
J. YÁÑEZ ET AL.
Fig. 8. Immunoreactivity to serotonin (5-HT) (A,B) and calretinin
(C,D) in the parapineal organ (A–C) and left habenula (D) of river
lamprey. A,B: Transverse sections through the rostral (A) and caudal
region (B) of the vesicle showing 5-HT-ir cells and fibers (arrowheads).
Asterisks, parapineal vesicle; star, parapineal ganglion. C: Sagittal
section through the pineal complex showing CR-ir cells in the parapi-
neal ganglion (star). Asterisk, parapineal vesicle. D: Sagittal section
through the left habenula showing groups of CR-ir cells in the rostral
part (open star) similar in appearance to those of the parapineal
ganglion. In C,D, rostral is at the right. Scale bars ⫽ 50 µm in A,B,
100 µm in C,D.
The present results involving connectivity and CR immunocytochemistry of the parapineal organ also support the
idea, based on ontogenetic (Studnicka, 1905) and electron
microscopic (Meiniel and Collin, 1971) studies, that the
ganglion is a part of the left habenular structure, which
undergoes a rostralward migration during development.
In this sense, the presence of parapineal afferent fibers
running in the left fasciculus retroflexus to the interpeduncular neuropil and of a bilateral efferent projection from
the habenulopetal subhippocampal-thalamic nucleus to
the parapineal organ are clearly habenular characteristics
(Yáñez and Anadón, 1994). Calretinin staining of the
neurons of the left habenula and the parapineal ganglion
is also similar. The nomenclature used by Studnicka
(accessory left habenular nucleus for the ganglion, and
habenular tract or habenular lamina for the laminar tract
that connects it with the left habenula), is very descriptive.
Despite this, the traditional terms adopted here (parapineal ganglion and tract) are less confusing for new readers.
Results seen after application of DiI to the left fasciculus
retroflexus or the left habenula indicate that the cells of
the parapineal vesicle do not project beyond the left
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
183
Fig. 9. ␥-Aminobutyric acid (GABA) immunoreactivity in the
parapineal organ of river lamprey. A: Sagittal section showing the
parapineal tract (solid arrow), ganglion (star), and vesicle (asterisk).
Numerous GABA-immunoreactive (GABA-ir) fibers are observed in
the ganglion, but some also pass to the parapineal vesicle. Note also
GABA-ir fibers running through the parapineal tract. Open arrow,
pineal stalk. B: Transverse section through the parapineal ganglion
(star) and vesicle (asterisk) showing GABA-ir fibers that also enter the
walls of the vesicle (arrowheads). C: Detail of GABA-ir terminals in
the parapineal ganglion. P, pineal organ. Scale bars ⫽ 50 µm in A,B, 10
µm in C.
habenula. The afferents of the parapineal vesicle of the
lamprey are similar to those of the parapineal organ of
trout (Yáñez et al., 1996) and the parietal (parapineal)
organ of lizards (Engbretson et al., 1981), which project
exclusively to the left habenula. The asymmetrical relationship of the parapineal nerve with the left habenula has
been reported in other reptiles by using nonexperimental
methods (Dendy, 1911; von Haffner, 1953; Ortman, 1960;
Kappers, 1965). All these parapineal organs of lamprey,
teleosts, and reptiles have also in common their develop-
ment from a rostral parietal vesicle (Hill, 1891; Nowikoff,
1910; Dendy, 1911; Meiniel and Collin, 1971; Corujo and
Anadón, 1986), whereas the pineal organs develop from
the posterior parietal vesicle of the embryo. The shared
parapineal-left habenula projection, together with the
same embryonic origin, are hard to explain if the parapineal appeared independently in these vertebrate groups,
which suggests that these parapineal organs have evolved
from a common precursor. The vesicular character of the
parapineal organ is also shared by lampreys, coelacanths,
184
J. YÁÑEZ ET AL.
Fig. 10. NPY-ir fibers in the parapineal organ (A,B), NPY-ir cells in
the subhippocampal nucleus (C), and galanin-ir fibers in the parapineal ganglion (D,E) of river lamprey. A: Sagittal section showing
NPY-ir fibers in the parapineal ganglion (star). Asterisk, parapineal
vesicle. Rostral is at the left. B: Transverse section showing NPY-ir
fibers in the parapineal ganglion (star). Asterisk, parapineal vesicle.
C: Horizontal section passing through the striatum and thalamus
showing numerous NPY-ir perikarya in the subhippocampal nucleus
(open arrows). Rostral is at the left. Arrow, external sulcus between
the telencephalic lobe and the thalamus. D,E: Transverse sections
through the parapineal ganglion (black stars) (D, rostral; E, caudal)
showing a few galanin-ir fibers (arrowheads). Asterisk, parapineal
vesicle; open star in E, pineal atrium. For abbreviations, see list. Scale
bars ⫽ 50 µm.
Sphenodon, and lizards. It is, however, puzzling why other
extant vertebrate groups (elasmobranchs, amphibians,
birds, and mammals) lack a parapineal organ and why it
has not been found in many bony fishes, although it is well
developed in the coelacanth Latimeria chalumnae (Hafeez
and Merhige, 1977). The hypothesis that all parapineal
organs have a common origin suggests that their absence
in some extant vertebrate groups must be attributable to
regressive changes that occurred multiple times independently. Although a regressive tendency of the parapineal in
benefit of the pineal might explain the scarce development
of this organ in the trout and other teleosts, or the
appearance during development of a vestigial rostral
parietal organ (pre-epiphysis) in some birds and mammals, which exceptionally can persist in adults (Beccari,
1943, pp. 363), whether early members of these groups
possessed this organ is not known. Because of its embryonic origin from a caudal parietal vesicle (Beccari, 1943)
and its connections of pineal type (Eldred et al., 1980), the
frontal organ of the frog does not appear to be a parapineal
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
organ. More studies are need to understand the phylogeny
of parapineal organs.
Parapineal photoreceptors
Immunocytochemistry carried out with antisera to opsins and other visual proteins indicates that the lamprey
parapineal vesicle is heterogeneous with respect to its
photoreceptor cells (Vigh-Teichmann et al., 1983; Kuo et
al., 1988; Tamotsu et al., 1990, 1994; Garcı́a-Fernández et
al., 1997), in agreement with results of earlier ultrastructural studies (Meiniel and Collin, 1971; Cole and Youson,
1982). Our results demonstrated that different photoreceptor populations are labeled with antisera to SP, ChAT, and
5-HT presenting important interspecific differences.
In sea lamprey, most SP-ir cells occupy different regions
of the vesicle to those of the cells labeled by DiI application
to the left habenula. Results from experiments using
immunocytochemistry and DiI tracing indicate that the
axons of most of these cells only extend as far as the
parapineal ganglion neuropil, only a few SP-ir fibers are
observed in the parapineal tract. Because of their bipolar
appearance, mainly dorsal and lateral location in the
vesicle, and the abundance of their processes in the
ganglion, SP-ir cells may correspond to the type II photoreceptors revealed by electron microscopy in this species
(Cole and Youson, 1982), although this should be investigated further. In the sea lamprey, SP-ir perikarya were
reported in a region of the corpus striatum, which may
include, in part, our subhippocampal nucleus, and SP-ir
fibers were observed in the ventral habenular nucleus
(Nozaki and Gorbman, 1986). The possibility that their
corpus striatum also contributes with a few SP-ir fibers to
the parapineal ganglion in the sea lamprey, thus, cannot
be ruled out. In the river lamprey, the antiserum to SP did
not reveal any cells in the parapineal organ, although a
few SP-ir fibers were seen to run through the parapineal
tract into the ganglion. These fibers were probably efferent. Differences between two species of lamprey (Petromyzon marinus and Entosphenus tridentatus) in both SP
immunoreaction staining and distribution of SP-ir perikarya in the brain was also previously reported (Nozaki
and Gorbman, 1986).
The presence of SP-ir structures has been described in
other parapineal organs. In trout, Ekström and Korf
(1986) claim that the parapineal organ receives a distinct
SP-ir innervation by means of the habenular nucleus.
Tracing studies of trout parapineal organs, however, found
no parapinealopetal neurons either in the habenula or
other brain regions whereas the parapinealofugal fibers
apparently extend to the same area of the left habenula
that is positive to SP (Yáñez et al., 1996). These contradictory results in trout can only be explained if the parapineal
perikarya were SP-ir and were, thus, the origin of the SP-ir
parapineal tract and neuropil in the dorsal left habenula.
This explanation would be in agreement with the lack of a
neuropil in the parapineal of trout as revealed by electron
microscopy (Rüdeberg, 1969). SP-ir fibers were also found
in both the parietal organ and left habenula of lizard
(Engbretson et al., 1982). Despite the parietopetal cells not
being able to be characterized, the SP-ir fibers in the
parietal organ were considered to be efferent because of
the lack of SP-ir perikarya in the parietal organ and left
habenula. Thus, all the parapineal organs studied appear
to be related with SP-ir systems. On the other hand, the
habenula of mammals (Ronnekleiv and Kelly, 1984; Con-
185
testabile et al., 1987), newt (Taban and Cathieni, 1983),
and Apteronotus, an electric fish (Weld and Maler, 1992)
contains SP-ir perikarya.
Our ChAT immunocytochemical results suggest the
presence of an intrinsic cholinergic system in the parapineal organ of river lamprey. ChAT-ir cells form two apparently separated groups, one in the rostrodorsal region and
the other in the ventrocaudal region. The bipolar shape of
the cells is compatible with them being photoreceptors.
These groups of ChAT-ir cells give rise to nerve terminals
in the vesicle and parapineal ganglion, and thus appear to
form part of local circuits. Although the distribution of the
rostral group of ChAT-ir cells is similar to that of 5-HT-ir
photoreceptors, they probably represent separated populations on the basis of the different thickness and shape of
cells and the different distribution of their fibers. ChAT-ir
receptors are also present in the pineal organ of lampreys
(Pombal et al., in press). ChAT-ir cells were observed in the
pineal organ, but not in the parapineal organ, of a teleost
(Ekström and Korf, 1986; Ekström, 1987b). However, the
ChAT-ir cells of the teleost pineal organ have been considered as interneurons.
The presence of 5-HT-ir photoreceptors in the pineal
complex of lamprey has been previously reported by Meiniel (1978, 1980) and by Tamotsu et al. (1990), who consider
these cells as modified (neuroendocrine) photoreceptors.
5-HT-ir cells are far more numerous in the parapineal
organ of river lampreys (Tamotsu et al., 1990; present
results) than in that of sea lamprey. Axons of 5-HT-ir cells
are scarce, branch only occasionally, and do not appear to
leave the parapineal organ, terminating near the parapineal vesicle surface or occasionally entering the ganglion,
which is in agreement with the photoneuroendocrine
hypothesis (Collin, 1971; Meiniel, 1980; Tamotsu et al.,
1990). The dorsorostral location of most 5-HT-ir cells of
river lamprey does not correspond to those cells labeled by
DiI application to the left habenula, nor has any 5-HT-ir
fiber been observed in the parapineal tract. Distribution of
5-HT-ir cells and fibers in the parapineal organ was also
very different from that observed for SP-ir elements,
suggesting that they form different systems. The salmon
parapineal may contain some 5-HT-ir elements (Ekström
and Ebbesson, 1989). It is, however, intriguing that the left
habenular nucleus of Oncorhynchus kisutch contains a
subnucleus of 5-HT-ir cells (Ekström and Ebbesson, 1988),
apparently located in a position similar to the area innervated by parapineofugal fibers in Oncorhynchus mykiss
(Yáñez et al., 1996). Ekström and Ebbesson (1988) suggested that they may represent displaced pineal photoreceptors but, if they were modified photoreceptors, comparison with our results in lamprey would suggest the
parapineal organ as a more probable source for these cells.
Unlike the parapineal organ of lamprey and salmon, no
immunoreactivity to 5-HT has been observed in the parapineal organ of a teleost, Gasterosteus aculeatus, at any
stage of development (van Veen et al., 1984).
The bipolar parapineal cells labeled by DiI application to
the left habenula form a population of long axon cells, as
was also indicated by Samejima et al. (1989) and Tamotsu
et al. (1994), who considered them as photoreceptors.
These cells had been labeled in the pineal and parapineal
organ of lamprey by application of horseradish peroxidase
to the ‘‘transected end of the pineal tract’’ (Samejima et al.,
1989; Tamotsu et al., 1994). Our DiI results in lamprey,
however, indicate that the targets of pineal and parapineal
186
J. YÁÑEZ ET AL.
long axon bipolar cells are different, the pineal cells not
being labeled from the left habenula, and the converse
being true for those of the parapineal organ. The long axon
cells of lampreys are roughly similar to bipolar cells
revealed in the trout pineal organ after horseradish peroxidase labeling of the proximal pineal stalk (Ekström,
1987a), although the targets of these cells are not known.
The ventral region of the parapineal organ of L. japonica
is characterized by the presence of cells immunoreactive to
rod-opsin, s-antigen, or visinin, thus differing from the
dorsal region (Tamotsu et al., 1990). For L. fluviatilis,
rod-opsin was also reported in only a circumscribed area of
the ventral region (Vigh-Teichmann et al., 1983). Because
in the two species studied here long axon cells occupy the
ventral region of the parapineal vesicle, they might correspond with cells containing one or more of these substances.
Parapineal efferents
An interesting result of the present investigation is that
the parapineal ganglion is innervated by pretectal and
telencephalic neuronal populations. Moreover, results of
immunocytochemistry indicate that the parapineal organ
receives at least two main types of efferent fiber through
the parapineal tract, which were GABA- and/or NPYimmunoreactive. GABA-ir fibers also enter the proximal
wall of the parapineal vesicle. This finding suggests that
the parapineal organ activity is modulated by these two
systems in lamprey. Comparison of distribution of GABAand NPY-ir brain nuclei with the results of application of
DiI to the parapineal organ, indicates that the NPY-ir
efferent fibers probably originate in the subhippocampalthalamic nucleus. Because GABA-ir cells were observed
both in the pretectal and subhippocampal nuclei, GABA-ir
efferent fibers might originate in either of these nuclei. It is
also conceivable that some GABA-ir efferent fibers in the
parapineal ganglion also may be NPY-ir, as are those found
in the saccus vasculosus system of trout (Yáñez et al.,
1997), but this does not appear to be the case for GABA-ir
fibers projecting to the vesicle. However, additional types
of efferent fibers immunoreactive to galanin (river lamprey), SP (river lamprey, sea lamprey?), and somatostatin
(sea lamprey, Yáñez et al., 1992) have been observed in
small numbers in the parapineal ganglion, and the list of
substances may increase with future studies.
The efferent innervation of the parapineal organ is
noticeable in the context of the pineal complex of other
vertebrates, but it is not if we consider that the parapineal
ganglion of lamprey has habenular characteristics. The
efferent system of the parapineal organ is notably similar
to that the habenulopetal system. In a previous study of
the larval sea lamprey habenula (Yáñez and Anadón,
1994), only a single habenulopetal nucleus, located in the
subhippocampal-thalamic nucleus, was reported. Neurons
of this nucleus, and of other telencephalic neurons, were
also labeled in adult river lamprey after DiI application to
the left habenula (present results). The main problem with
interpretation of the telencephalo-habenular innervation,
however, was to know which telencephalic fibers running
through the habenular commissure are en passant fibers
and that, in fact, connect with the habenula (Northcutt
and Puzdrowski, 1988; Yáñez and Anadón, 1994). This was
not a problem with the parapineal organ because of its
terminal position. The application of DiI to the parapineal
organ labeled a single nucleus in the telencephalon, the
subhippocampal-thalamic nucleus. This type of application showed fewer bilateral labeled neurons in the subhippocampal-thalamic nucleus than the application to the
habenula (Yáñez and Anadón, 1994; present results),
suggesting that many neurons of the subhippocampalthalamic nucleus only have projections to the habenula.
After DiI application to the parapineal organ, labeled
fibers were observed to run rostralward to the primordium
hippocampi (Johnston, 1902; Heier, 1948). Our results
suggest that they are collateral branches of axons of
neurons of the subhippocampal-thalamic nucleus. Further
evidence that the cells of the subhippocampal-thalamic
nucleus may project simultaneously to the olfactory bulb
and the parapineal organ comes from two in vitro experiments of application of biotinylated dextran amine to the
olfactory bulb (M.A. Pombal, unpublished data). These
experiments labeled both neurons in this nucleus and
efferent fibers in the parapineal organ that were similar to
those revealed by GABA and NPY immunocytochemistry.
In a similar way, cells of the main habenulopetal telencephalic nucleus of trout (entopeduncular nucleus) appear to
project both to the habenula and the lateral telencephalon
(Yáñez and Anadón, 1996). The different position of the
entopeduncular-telencephalic projection in trout may be
due to the telencephalic eversion occurring in teleosts
(Northcutt, 1995). The characteristics shared by the subhippocampal-thalamic nucleus of lamprey and the entopeduncular nucleus of trout are more easily explained assuming that they are homologous structures than if they have
appeared independently. These nuclei would relay information from basal telencephalic systems to both the habenula
and dorsal telencephalon (for a comparison of the entopeduncular nucleus of trout with habenulopetal nuclei of
other vertebrates, see Yáñez and Anadón, 1996).
Moreover, the application of DiI to the parapineal organ
revealed a pretecto-parapinealopetal (and habenulopetal)
system. Because DiI application to the pretectum led to
fibers in both habenulae being labeled, this nucleus appears to have been overlooked in a previous study of larval
lamprey habenula (Yáñez and Anadón, 1994), probably
because of the very short distance from this nucleus to the
application point, the poor development of the pretectal
region in larvae, or both. The cells of the parapinealopetal
(and habenulopetal) pretectal nucleus extend dendrites
through a pretectal region which in lamprey receives both
primary visual and pineal fibers (Vesselkin et al., 1980; De
Miguel et al., 1990; Puzdrowski and Northcutt, 1989;
Yáñez et al., 1993). These results, thus, suggest that
activity of the lamprey parapineal ganglion and vesicle is
under control of two other photoreceptive organs, the
pineal organ and the retina, that may modulate the input
of the parapineal organ on the habenulo-interpeduncular
system by means of this nucleus.
Parapineal-interpeduncular projection
One of the most interesting findings of our study is the
afferent projection from the parapineal ganglion to the
interpeduncular nucleus by means of the left fasciculus
retroflexus, as well as the close relationship between the
parapineal vesicle on one side and both this ganglion and
the left habenula on the other. DiI application to the
parapineal organ produces labeling of this fascicle which is
rather similar to that previously reported after habenular
labeling (Yáñez and Anadón, 1994). Unlike the habenula,
the ganglion only has projections to the rostral (isthmic)
PARAPINEAL ORGAN CONNECTIONS IN LAMPREY
portion of the interpeduncular complex, traditionally included in the mesencephalon (Heier, 1948), whereas habenular fibers also run in the rhombencephalon proper up
to the level of the trigeminal nerve. The caudal habenular
fibers of lamprey might be comparable to habenular fibers
ending in the raphe and dorsal tegmental nucleus in trout
(Yáñez and Anadón, 1996) and lizard (Dı́az and Puelles,
1992). Because the parapineal ganglion and all portions of
the habenular nucleus of lamprey have projections to the
interpeduncular nucleus (present results; Yañez and Anadón, 1994), they can only be compared with the medial
habenular nucleus of mammals (Herkenham and Nauta,
1977; Contestabile and Flumerfelt, 1981).
Functional considerations
The ‘‘parapineal organs’’ of three different groups of
vertebrates (lampreys, teleosts, and lizards) share a similar relationship with the habenulo-interpeduncular system (see above), a system which appears to be one of the
most constant in all vertebrates (Herrick, 1948). In mammals, the habenulo-interpeduncular system is involved in
limbic functions, including autonomic and endocrine control mechanisms, and aspects of sexual and defensive
behavior (Herkenham and Nauta, 1979; Sutherland, 1982;
Glaser and Barfield, 1984; Meszaros et al., 1985). However, practically nothing is known about the functions of
this system in non-mammals. Herrick (1948) suggested
that this system is the principal component that controls
feeding behavior in ‘‘lower’’ species, mediating its integration with olfactory activity. Our anatomical results in
lampreys do not contradict this view, but unfortunately,
there are no functional studies of the habenulo-interpeduncular system in lampreys or other nonmammalian vertebrates. Experiments in lizard indicate that the parietal eye
is responsive to light (Miller and Wolbarsht, 1962; Dodt
and Scherer, 1968; Engbretson and Lent, 1976), being
modulated by the pineal gland (Engbretson and Lent,
1976). However, the parietal eye plays little or no role in
maintenance of circadian melatonin rhythm (Underwood
and Calaban, 1987), circadian electroretinogram rhythm
(Shaw et al., 1993), daily thermoregulatory behavior (Tosini
and Menaker, 1996), or the locomotor rhythm (Innocenti et
al., 1993), all these rhythms being affected by pineal
manipulation. Experimental studies on possible functions
of the pineal complex in lamprey (Joss, 1973a,b; Cole and
Youson, 1981) have not distinguished between the pineal
and the parapineal organs. Thus, the role played by the
strikingly asymmetrical projection of parapineal organs in
the left habenulo-interpeduncular system is presently a
mystery that must wait to be resolved by future studies.
CONCLUSIONS
The major conclusion of this study is that the parapineal
organ of lamprey is a part of the afferents to the habenulointerpeduncular system, both being specifically connected
to the left habenula and innervated by efferents from the
two main habenulopetal nuclei (i.e., the subhippocampalthalamic and pretectal nuclei). The parapineal vesicle is
mainly connected to the parapineal ganglion by short axon
cells, but also has long projections to the left habenula.
Both the vesicle and the ganglion possess cell populations
and fibers that are heterogeneous with respect to neurotransmitters, neurotransmitter synthesizing enzymes, neuropeptides, and a calcium binding protein, some of which
187
(SP, ChAT, galanin, CR) have been found in the lamprey
parapineal for the first time. Finally, this study proposes a
general distinction between pineal and parapineal organs
of vertebrates on the basis of the ontogeny and the very
specific relation of ‘‘true’’ parapineal organs with the left
part of the habenulo-interpeduncular system.
ACKNOWLEDGMENTS
The authors thank Prof. S. Grillner (Karolinska Institute) for providing river lamprey brains and laboratory
facilities to one of us (M.A.P.), and A. González and O.
Marı́n (University Complutense of Madrid) for helping
with ChAT immunocytochemistry. We also thank Dr. Christine Francis for linguistic revision. This research was
supported by Spanish Education Ministry Grant PB96–
0945-C03 to R.A. and J.Y.; by Xunta de Galicia Grants
XUGA20012B96 to M.A.P. and XUGA20002B97 to R.A.
and J.Y.; and by University of Vigo Grant 64102C734 to
M.A.P.
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