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Afferents of the lamprey striatum with special reference to the dopaminergic system A combined tracing and immunohistochemical study

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THE JOURNAL OF COMPARATIVE NEUROLOGY 386:71–91 (1997)
Afferents of the Lamprey Striatum With
Special Reference to the Dopaminergic
System: A Combined Tracing and
Immunohistochemical Study
MANUEL A. POMBAL,1,2 ABDELJABBAR EL MANIRA,1 AND STEN GRILLNER1*
Nobel Institute of Neurophysiology, Department of Neuroscience, Karolinska Institutet,
S-17177, Stockholm, Sweden
2Departamento de Biologı́a Fundamental, Facultad de Ciencias, Universidad de Vigo,
E-36200, Vigo, Spain
1The
ABSTRACT
The origin of afferents to the striatum in lamprey (Lampetra fluviatilis) was studied by
using fluorescein-coupled dextran-amines (FDA). Injection of FDA into the striatum retrogradely labeled several cell populations in the forebrain and the rostral rhombencephalon. No
retrograde labeled cells were seen in the mesencephalon.
A dopamine-specific antiserum was used to determined the distribution of dopaminergic
perikarya and fibers. Many dopamine-immunoreactive (DA-ir) fibers were present throughout
the brain, but the highest density of labeled fibers was in the mediobasal prosencephalon,
especially in the striatum, the lateral hypothalamic area, and the neurohypophysis. Most
DA-ir cells were located in the mediobasal diencephalon (preoptic region, nucleus commissurae postopticae, hypothalamus, and nucleus tuberculi posterioris). In the mesencephalon,
only a few immunopositive cells were observed in the tectum opticum. In the rhombencephalon, DA-ir cells were observed in the isthmic region, dorsally to the descending trigeminal
tract, and caudally to the posterior rhombencephalic reticular nucleus. The rostralmost spinal
cord received many descending DA-ir fibers from the brainstem. Along the spinal cord, DA-ir
neurons were also found, some of which projected to the medioventral surface, forming a
prominent plexus. On the basis of double-labeling experiments, it is shown that the
dopaminergic input to the striatum originates from the nucleus tuberculi posterioris.
Thus, the striatum receives inputs from different structures, including a strong dopaminergic innervation from the diencephalon. Much of the dopaminergic system in Lampetra
fluviatilis is basically similar to that seen in some teleosts, but it presents differences with
other anamniote (elasmobranch) as well as amniote groups. J. Comp. Neurol. 386:71–91,
1997. r 1997 Wiley-Liss, Inc.
Indexing terms: Lampetra fluviatilis; telencephalon; basal ganglia; fluorescent tracers; dopamine
The vertebrate basal ganglia play an important role in
the control of different types of motor behavior. Comparatively little is known about the organization of the basal
ganglia in the lamprey, a vertebrate that separated from
the main vertebrate evolutionary line 450 million years
ago (Hotton, 1976). The lamprey nervous system is of
interest not only from an evolutionary perspective but also
because more knowledge is available concerning the neuronal circuitry in the brainstem-spinal cord than for most
other vertebrates (for reviews, see Rovainen, 1979; Brodin
and Grillner, 1990; Grillner et al., 1995).
The striatum is one of the most prominent cell masses in
the lamprey telencephalon medium (Johnston, 1912; Her-
r 1997 WILEY-LISS, INC.
rick and Oberchain, 1913; Heier, 1948). The striatum is
composed of several layers of closely packed cells separated from the ependyma by a rather broad neuropil
(Johnston, 1902; Heier, 1948). The telencephalon of lampreys consists of two well-developed, evaginated, dorsolat-
Grant sponsor: Swedish Medical Research Council; Grant number: 3026,
Grant number: 11562.
*Correspondence to: Prof. Sten Grillner, The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, S-17177
Stockholm, Sweden. E-mail: sten.grillner@neuro.ki.se
Received 18 July 1996; Revised 17 April 1997; Accepted 22 April 1997
72
M.A. POMBAL ET AL.
eral olfactory bulbs and cerebral hemispheres and an
unevaginated telencephalon medium. Homologies between telencephalic cell groups in lampreys and other
vertebrates are difficult to determine, but several pallial
subdivisions have been proposed based on their topography and secondary olfactory afferents (Northcutt and
Puzdrowski, 1988). Recent experimental studies on the
connections of the lamprey telencephalon (Northcutt and
Puzdrowski, 1988; Polenova and Vesselkin, 1993; Northcutt and Wicht, 1997) have focused on secondary and
tertiary olfactory projections and major pallial connections. However, no studies have been performed on the
connections of the subpallial structures.
The limited information available concerning afferents
and efferents of the lamprey striatum (Johnston, 1902;
Heier, 1948) are based mainly on general and silver
stainings. The striatum in lamprey has a chemical organization similar to that of other vertebrates, with neurons
that are immunoreactive towards g-aminobutryic acid
(GABA) and substance P and that are positive for acetylcho-
line esterase (Pombal et al., 1995). In addition, it is also
densely innervated by fibers that are immunoreactive to
serotonin (5-HT; Brodin et al., 1988, 1990b; Pierre et al.,
1992), substance P (Nozaki and Gorbman, 1986; Pombal et
al., 1995), histamine (Brodin et al., 1990a), neurotensin
(Brodin et al., 1990b), tyrosine hydroxylase (TH; Pierre et
al., 1994), galanin (Jiménez et al., 1996), and enkephalin
(Pombal et al., 1995).
In mammals, the dorsal striatum is involved in sensorimotor and cognitive functions, whereas the ventral striatum is implicated in the processing of limbic information
entering the basal ganglia (Alheid et al., 1990). A wide
variety of sources of inputs to the striatum and nucleus
accumbens have been described, with both the striatum
and the nucleus accumbens receiving a very dense dopaminergic innervation (Parent, 1986; Smeets and Reiner,
1994b; Medina and Reiner, 1995), but the number of
known efferent connections is more limited (Alheid et al.,
1990).
Abbreviations
aon
arn
b
c
ca
cc
cgl
ch
cp
cpi
cpo
cpr
cpt
cr
cs
ctp
D
dcf
dh
dhy
dm
dmc
dn
dp
dth
dV
f
go
h
hy
igl
III
ip
IV
lc
lco
lpd
lpv
M
M3
M5
mlf
mn
mp
mra
mrn
mt
nb
nca
ncp
ncpi
anterior octavomotor nucleus
anterior rhombencephalic reticular nucleus
commissura interbulbaris
cerebellar plate or cerebellar commissura
commissura anterior
central canal of the spinal cord
corpus geniculatum laterale
chiasma opticum
commissura posterior
commissura postinfundibularis
commissura postopticae
commissura praeinfundibularis
commissura posttectalis
cell region of the spinal cord
central stratum (tectum opticum)
commissura tuberculi posterioris
diencephalon
dorsal column fibers
dorsal horn of the spinal cord
nucleus dorsalis hypothalami
dorsomedial telencephalic neuropil
dorsomedial column of the spinal cord
dorsal nucleus of the octavolateral area
dorsal pallium
nucleus dorsalis thalami
radix sensitivus nervi trigemini
fasciculus retroflexus
glomeruli olfactorii
ganglion habenulae
hypothalamus
internal granular layer of the olfactory bulb
nucleus nervi oculomotorii
nucleus interpeduncularis
nucleus nervi trochlearis
lateral column of the spinal cord
locus coeruleus
dorsal part of the lateral pallium
ventral part of the lateral pallium
mesencephalon
third Müller cell
nucleus M5 of Schober
medial longitudinal fasciculus
medial nucleus of the octavolateral area
medial pallium
mesencephalic reticular area
medial rhombencephalic reticular nucleus
mesencephalic tegmentum
nucleus of Bellonci
nucleus commissurae anterior
nucleus commissurae posterioris
nucleus commissurae postinfundibularis
ncpo
ncpr
nh
nmlf
noa
np
npo
nrpo
ntp
nts
ob
os
ot
P
p
pch
PP
prn
Ps
ps
R
ra
ri
rpo
rpos
rpro
s
SC
sd
slf
slh
so
ss
st
T
to
ts
vc
vd
vhy
VII
vla
vlp
Vm
vm
vn
vr
vt
vth
X
nucleus commissurae postopticae
nucleus commissurae praeinfundibularis
neurohypophysis
nucleus fasciculi longitudinalis medialis
nucleus olfactorius anterior
nucleus praetectalis
nucleus praeopticus
nucleus recessus postopticus
nucleus tuberculi posterioris
nucleus tractus solitarii
olfactory bulb
optic stratum (tectum opticum)
tectum opticum
pineal organ
ventromedial plexus of the spinal cord
plexus choroideus
parapineal organ
posterior rhombencephalic reticular nucleus
pineal stalk
periventricular stratum (tectum opticum)
rhombencephalom
raphe region
recessus infundibuli
recessus postopticus
recessus posterior
recessus praeopticus
nucleus septi
spinal cord
saccus dorsalis
spinal lemniscal fibers
sulcus limitans of His
subcommisural organ
superficial stratum (tectum opticum)
striatum
telencephalon
tractus opticus
torus semicircularis
ventral column of the spinal cord
ventriculus diencephali
nucleus ventralis hypothalami
nucleus motorius nervi facialis
ventriculus lateralis anterior telencephali
ventriculus lateralis posterior telencephali
nucleus motorius nervi trigemini
ventriculus mesencephali
ventral nucleus of the octavolateral area
ventriculus rhombencephali
ventriculus medius telencephali
nucleus ventralis thalami
nucleus motorius nervi vagi
STRIATAL AFFERENTS IN LAMPREY
Dopamine (DA), together with adrenaline and noradrenaline, are the major catecholamines used by the nervous
system, with TH as the key enzyme involved in their
biosynthesis. Current understanding of catecholaminergic
systems in the lamprey forebrain is based mainly on
studies of the distribution of monoamine oxidase (MAO;
Tsuneki et al., 1975) and formaldehyde-induced histofluorescence (FIF; Honma, 1969; Honma and Honma, 1970;
Baumgarten, 1972; Konstantinova, 1973; Ochi and Hosoya, 1974; Tsuneki et al., 1975) and on immunohistochemical markers, such as noradrenaline (Steinbusch et al.,
1981b), TH (Brodin et al., 1990a; Pierre et al., 1994), and
aromatic amino acid decarboxylase (AADC; Mahouche et
al., 1994). Most of these studies have focused on the
hypothalamohypophyseal system. Catecholaminergic terminals in the striatum were initially described in the river
lamprey by Baumgarten (1972), and, more recently, immunohistochemical studies have revealed the presence of
TH-immunoreactive (ir) fibers (Pierre et al., 1994).
Since the development of antibodies against DA (Geffard
et al., 1984), many studies have been carried out in
different groups of vertebrates (for review, see Smeets and
Reiner, 1994a). Some preliminary results with DA antibodies were also reported in larval (Yáñez et al., 1992) and
adult (Pierre et al., 1994) lampreys. In lampreys, a small
catecholaminergic cell group suggested to be a possible
homologue of the substantia nigra in amniotes has been
described in the region of the posterior tubercule (Baumgarten, 1972), which is incorporated in the base of the
midbrain in tetrapods. In the spinal cord, two populations
of DA-ir cells have recently been described, cerebrospinal
fluid (CSF)-contacting cells around the central canal and
5-HT/DA-containing cells, which form a ventromedial
plexus (Schotland et al., 1995, 1996).
The aim of the present study was threefold: 1) to
describe the input to the striatum from different forebrain
structures by injection of the tracer fluorescein-coupled
(FDA) and biotinylated (BDA) dextran-amines, 2) to reveal
the distribution of dopaminergic cells and fibers in the
lamprey forebrain and brainstem by using antibodies
against DA, and 3) to determine the cells of origin of the
dopaminergic innervation of the lamprey striatum by
using double-labeling experiments with BDA and a TH
antiserum. Part of these results have been published in
abstract form (Pombal et al., 1995, 1996).
MATERIALS AND METHODS
Animals
This study was performed on 38 adult (20–35 cm in total
length) river lampreys (Lampetra fluviatilis L.) that were
captured in Söderhamn, Sweden and kept in aerated
freshwater aquaria at 4–10°C. Fourteen animals were
used for fluorescence-tracing studies, 16 for immunocytochemistry, and eight for double labeling. Prior to experimentation, the animals were anesthetized with 0.01% solution
of tricaine methanesulphonate (MS-222; Sandoz, Basel,
Switzerland) in fresh water. The protocols used in this
study were approved by the ethical committee of animal
experiments (Stockholm, Sweden).
In vitro fluorescence-tracing experiments
Animals were decapitated, and the brains were dissected out in a cooled physiological Ringer’s solution with
the following composition (in mM): 138 NaCl, 2.1 KCl, 1.8
73
CaCl2, 1.2 MgCl2, 4 glucose, 0.5 L-glutamine and 2 Hepes.
The solution was bubbled with 100% O2 for 15 minutes,
and the pH was adjusted to 7.4 with NaOH. The pineal and
parapineal organs and the plexus choroideus were removed. The commissura interbulbar and, sometimes, the
commissurae habenular and posterior were cut to facilitate access to the impar telencephalic ventricle. FDA
(3,000 MW; Molecular Probes, Eugene, OR) paste (a concentrated solution of the tracer in distilled water that was
desiccated on the tip of a 000 insect pin) was applied to the
brains at the level of the striatum region for 1–2 minutes.
The brains were subsequently washed to remove the
excess tracer and kept at 4°C in the same Ringer’s solution
for 24–65 hours to allow transport of the FDA. The brains
were then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 24–96 hours, washed in PB,
and treated for 30 minutes with 1% sodium borohydride in
the same buffer. After washing with 0.1 M PB, pH 7.4, and
cryoprotection with 15% and 30% sucrose solutions in the
same buffer, the brains were mounted in OCT (Optimal
Cutting Temperature; Miles Inc., Elkhart, IN), frozen with
CO2, and transversely sectioned at 30 µm by using a Leitz
1720 digital cryostat. Sections were then collected and
mounted on gelatin-coated slides and were examined and
photographed with a Nikon microphot-FXA microscope.
Immunocytochemical procedure
Animals were decapitated, and the brains were quickly
removed in physiological Ringer’s solution and fixed overnight at 4°C in 4% glutaraldehyde (GTA) in 0.1 M PB, pH
7.4. The brains were subsequently washed in PB (3 3 10
minutes) and treated for 30 minutes with 1% sodium
borohydride to reduce Schiff bases caused by GTA fixation.
Transversal, sagittal, or horizontal cryostat sections (16
µm thick) were obtained after cryoprotection with 30%
sucrose in 0.1 M PB. A specific rabbit DA antiserum
(Steinbusch et al., 1991) and three different immunostaining protocols (peroxidase-antiperoxidase: PAP, n 5 10;
streptavidin-biotin: SAB complex, n 5 4, and fluorescence,
n 5 2) were used.
For the PAP or SAB complex, all sections were treated
for 20–30 minutes with 0.3% H2O2 to abolish pseudoperoxidase activity in erythrocytes. Sections were washed in 0.1
M phosphate-buffered saline (PBS; pH 7.4) and subsequently incubated in a humid chamber. For the SAB
complex protocol, sections were incubated at 4°C overnight
with the DA antiserum raised in rabbit (Steinbusch et al.,
1991; dilution: 1:500 to 1:1,000), rinsed in PBS (3 3 10
minutes), incubated for 30 minutes at room temperature
with biotinylated goat anti-rabbit IgG (Dakopatts, Glostrup, Denmark; diluted 1:200), rinsed with PBS, and
incubated again with StreptABComplex/horseradish peroxidase (HRP; Elite, ABC kit; Vector Laboratories, Burlingame, CA) for 30 minutes. Following extensive rinsing, the
complex was developed by incubation in 3,38-diaminobenzidine (Sigma, Stockholm, Sweden; 50 mg/100 ml) and
0.003–0.01% hydrogen peroxide for 8–10 minutes. For the
PAP protocol, sections were incubated sequentially by
using the same DA antiserum (dilution 1:800 to 1:1200) at
4°C overnight, goat anti-rabbit IgG (Dakopatts; diluted
1:60) for 1 hour at room temperature, and rabbit PAPcomplex (Dakopatts; diluted 1:300) for 45 minutes; the
complex was developed as described above. Sections were
then rinsed thoroughly, dehydrated in graded alcohols,
and coverslipped.
74
M.A. POMBAL ET AL.
In experiments conducted by using the indirect immunofluorescence method, the sections were incubated with
rabbit DA antiserum (Steinbusch et al., 1991; dilution
1:400 to 1:600) for 18–24 hours at 4°C, rinsed in PBS, and
then incubated for 1 hour at room temperature with
fluorescein isothiocyanate (FITC)-coupled goat anti-rabbit
antibodies (Boehringer-Mannheim, Germany; dilution 1:40
to 1:80). After being rinsed in PBS, the sections were
mounted in a glycerol-PBS mixture (3:1).
All sections were examined and photographed in a
Nikon microphot-FXA microscope. The different immunostaining procedures (PAP, SAB complex, and fluorescence)
produced the same pattern of DA immunoreactivity, although some cell populations showed stronger labeling
when using the SAB complex than when using immunofluorescence or PAP staining.
Double-labeling experiments
For double-labeling experiments, we used the same
procedure as described by Marı́n et al. (1995). Briefly, BDA
(3,000 MW; Molecular Probes Europe, Amsterdam, Netherlands) was applied in the striatum (as described above for
FDA). After survival times of 60–65 hours, the brains were
fixed overnight in 4% paraformaldehyde in 0.1 M PB, pH
7.4, cryoprotected with 30% sucrose, and transversally,
horizontally, or sagitally sectioned at 16 µm. Sections were
then incubated for 48 hours at 4°C with a rabbit TH
antiserum (Markey et al., 1980; dilution 1:400 to 1:600).
They were subsequently incubated with an FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch
Lab., Inc., West Grove, PA; dilution 1:60) and a Texas-redconjugated streptavidin complex (Vector Laboratories; dilution 1:100) to visualize TH and BDA, respectively. The
sections were mounted with a glycerin-PB medium, coverslipped, examined, and photographed with a Nikon microphot-FXA microscope. In some cases (four brains), BDA
was visualized by using a StreptABComplex/HRP (Elite,
ABC kit; Vector Laboratories) for 60 minutes and was
subsequently developed by incubation in 3,38-diaminobenzidine (Sigma; 50 mg/100 ml) and 0.003–0.01% hydrogen
peroxide for 8–10 minutes.
Nomenclature
For the forebrain and the mesencephalic areas, we have
used the anatomical nomenclature of Heier (1948) and
Schober (1964) and, for the pallial subdivisions, that of
Northcutt and Puzdrowski (1988). For the rhombencephalon, we have followed Nieuwenhuys (1977), Koyama et al.
(1989), and Ronan and Northcutt (1990). The subdivisions
of the spinal cord are modified from Van Dongen et al.
(1985).
RESULTS
The distribution of labeled cell bodies revealed by injections of FDA or BDA into the striatum will be described
first. It is demonstrated that the striatum receives inputs
from most telencephalic nuclei (cf. Fig. 1), including all
major divisions of the pallium. The remaining afferents
have a diencephalic or rhombencephalic origin and include
different thalamic and hypothalamic nuclei, the nucleus
tuberculi posterioris, the presumed locus coeruleus, and
the raphe region. A detailed description of the dopamine
immunoreactivity in the whole lamprey brain and spinal
cord will then be described. A very dense dopaminergic
innervation of the lamprey striatum is demonstrated as
well as the presence of a well-developed dopaminergic
system.
Afferent projections to the striatum
To characterize the cell populations projecting to the
striatum, FDA or BDA was injected into this nucleus (see
Materials and Methods). The locations of retrogradely
labeled fibers and cells after these injections are shown
schematically in transversel sections from different levels
(Fig. 1) and are summarized in a midsagittal projection
(Fig. 2). All striatal injections (st; see Fig. 1B) included
both the periventricular neuropil and the layer of cell
bodies underneath. After these injections, numerous labeled fibers were observed in the dorsal, lateral, and
ventral areas adjacent to the injection site. Retrogradely
labeled neurons occurred immediately rostral in the
nucleus commissurae anterior (nca; Figs. 1A, 2) and the
nucleus septi (s; Figs. 1A, 2) and, ventrally, in the nucleus
praeopticus (npo; Figs. 1B, 2). Some fluorescent cells were
also present in the dorsal pallium (dp; Figs. 1B,C, 2) and in
the ventral part of the medial pallium (mp; Figs. 1C, 2).
Larger numbers of retrogradely labeled cells were seen
both laterally in the internal granular layer of the olfactory bulb (igl; Figs. 1A,B, 2) and caudolaterally in the
ventral part of the lateral pallium (lpv; Figs. 1B,C, 2, 3A).
A few cells were also retrogradely labeled in the dorsal part
of the lateral pallium (lpd; Figs. 1B,C, 2). Caudal to the
striatum, labeled fibers coursed in the ventral and ventrolateral directions. Labeled fibers running ventrally divided into a medial and a ventral bundle (Fig. 1C). The
medial bundle decussated in the commissura postopticae
(cpo; Fig. 1C) and turned either dorsally toward the
contralateral nucleus ventralis thalami (vth) or ventrally
toward the hypothalamus (dhy and vhy), where some
retrogradely labeled cells were seen (Figs. 1D,E, 2). The
ventral bundle reached the nucleus commissurae postopticae (ncpo; Fig. 1C), the nucleus recessus postopticus (nrpo;
Fig. 1C), and the nucleus commissurae praeinfundibularis
(ncpr; Fig. 1F). Most of the remaining labeled fibers
reached the caudal hypothalamus, the nucleus ventralis
thalami, and the caudalmost diencephalon (Figs. 1D–F,
3E). Numerous cells were retrogradely labeled in the
nucleus dorsalis thalami in a subhabenular position (dth;
Figs. 1E, 2, 3B), the nucleus ventralis thalami (vth; Figs.
1D,E, 2, 3B,C), and the nucleus dorsalis and ventralis
hypothalami (dhy and vhy; Figs. 1D,E, 2, 3D,E). Ipsi- and
contralaterally labeled cells in the nucleus ventralis
thalami were located in both a subependymal and a
laterally displaced area. The subependymal cells were
small, elongated in shape, and located perpendicular to the
ventricular surface, whereas the laterally located cells
were bigger and polygonal (Fig. 3C). Labeled cells in the
hypothalamic areas were of both CSF- and non-CSFcontacting types (Fig. 3D,E). Some retrogradely labeled
cells also occurred within the nucleus tuberculi posterioris
(ntp; Figs. 1F, 2, 3D; cf. also Fig. 9B) in the basal
diencephalon. These cells were small and were either
round or pear-shaped. A few of them were located in the
midline or slightly contralateral to the injection site. Some
retrogradely labeled cells were also present in the rostral
rhombencephalon in both the isthmus region, constituting
the presumed locus coeruleus (lc; Fig. 1G; cf. also Fig. 9D),
and another area close to the midline below the trigeminal
Fig. 1. A–H: Diagrammatic representation of the distribution of
retrogradely labeled cell bodies (large dots) and fibers (dashed lines
and small dots) in transverse sections of the river lamprey brain
following a unilateral injection of fluorescein-coupled dextran-amines
(FDA) into the left striatum. The black oval region in B indicates the
site of the tracer injection, and the shaded area shows the subsequent
extent of its diffusion. On the left of A–H, short dashed lines represent
fiber tracts, and long dashed lines delineate the different cell populations. The sections are arranged from rostral to caudal, and the level of
each section is shown in a sagittal view of the brain in Figure 2. For
abbreviations, see list. Scale bar 5 1 mm.
76
M.A. POMBAL ET AL.
In the telencephalon proper, the distribution of DA-ir
fibers was most pronounced in the basal portion, particularly in the periventricular neuropil of the striatum (Figs.
4C,D, 5B, 6C,D). Numerous DA-ir fibers were also present
in the nucleus septi, the nucleus olfactorius anterior, the
nucleus commissurae anterior (Fig. 4B), and the ventral
part of the lateral pallium (Fig. 4C,D). In contrast, only a
few labeled fibers were seen in the dorsal part of the lateral
pallium (Fig. 4D), medial pallium (Fig. 4C–E), and dorsal
pallium (Fig. 4C,D).
Diencephalon
Fig. 2. Sagittal view of the river lamprey brain showing the
summed distribution of labeled cell bodies (dots) following a unilateral
injection of FDA into the striatum (st). Levels A–H illustrate the
relative positions of the transverse sections shown in Figure 1. For
abbreviations, see list. Scale bar 5 1 mm.
motor nucleus, forming part of the raphe region (Figs. 1H,
3F).
DA immunocytochemistry
In all vertebrates studied thus far, the striatum and the
nucleus accumbens are densely innervated by DA-ir fibers
originating mostly from the substantia nigra and the
ventral tegmental area (see Parent, 1986; Smeets and
Reiner, 1994b; Medina and Reiner, 1995). By using a
specific DA antibody, several groups of DA-ir neurons were
found in the lamprey (cf. Figs. 4, 5). The most prominent
populations were located in the diencephalon, whereas
fewer DA-ir populations were located in the telencephalon,
mesencephalon, and rhombencephalon.
The major anatomical localization of DA-ir perikarya
and fibers in L. fluviatilis is illustrated schematically in
selected transverse sections through the brain and spinal
cord (Fig. 4) and projected onto a midsagittal plane (Fig. 5).
A detailed description of the dopaminergic system is
reported below for each of the major brain subdivisions
(i.e., telencephalon, diencephalon, mesencephalon, and
rhombencephalon) and also in the spinal cord.
Telencephalon
Perikarya. The internal granular layer of the olfactory bulb contained numerous scattered and weakly labeled dopaminergic cell bodies (igl; Figs. 4A–C, 5, 6A).
These cells were small (5–12 µm in diameter) and round or
ovoid shaped. Round or elongated DA-ir cell bodies were
also found in the nucleus commissurae anterior (nca; Figs.
4B, 5, 6B). Some of these dopaminergic cell bodies had a
short apical process that reached and contacted the recessus praeopticus (rpro).
Fibers. Some short labeled fibers were found in the
olfactory bulb that originated from dopaminergic cells of
the internal granular layer (igl; Figs. 4A–C, 6A). The
dorsomedial telencephalic neuropil was also innervated by
DA-ir varicosities, presumably originating from the same
DA-ir neurons (Figs. 4C,D, 6A).
Perikarya. The medioventral part of the diencephalon
contained most of the DA-ir cell bodies in the brain of adult
L. fluviatilis (Figs. 4C–G, 5A, 7). The rostralmost DA-ir
neurons were located around the chiasma opticum, in the
region of the nucleus praeopticus (npo; Figs. 4C, 5A,
6B,C,E). This nucleus contained immunoreactive fusiform
cell bodies (approximately 10 3 25 µm in size) arranged
perpendicularly to the optic fibers. Most of these DA-ir
cells were located slightly dorsal to the optic fibers and
possessed short CSF-contacting processes that reached
and protruded into the recessus praeopticus, whereas
round and dorsally located cells had no CSF-contacting
processes. Caudal to this region, some DA-ir cells in the
nucleus commissurae postopticae (ncpo; Figs. 4D, 5A,
6B,E) formed a sparse population of small (10–15 µm),
round or slightly fusiform cells near the ventricular surface.
The most conspicuous population of DA-ir cell bodies
was found in the caudal hypothalamic walls of the diencephalic ventricle. The nucleus dorsalis and ventralis hypothalami (dhy and vhy; Figs. 4E,F, 5A, 6E, 7B,C) as well as
the nucleus commissurae postinfundibularis (ncpi; Figs.
4G, 5A, 7C–E) contained numerous DA-ir cell bodies
arranged in one to three rows beneath and among the
ependymal cells, parallel to the recessus infundibuli and
posterior. These cells were mostly bipolar, but some pearshaped perikarya were also seen in the ependymal layer.
Most, if not all, of these cells had apical CSF-contacting
processes that reached and protruded into the diencephalic (third) ventricle and long basal processes that
coursed to the ventrolateral neuropil, forming a dense
immunoreactive plexus (Figs. 4E,F, 6A,B). Some weak
DA-ir cells were also located in the nucleus tuberculi
Fig. 3. Photomicrographs of several transverse sections through
the brain of the river lamprey illustrating retrogradely labeled cell
bodies and fibers following biotinylated dextran amine (BDA; A,B,F) or
FDA (C–E) injection into the left striatum. A: Retrogradely labeled
cells (arrowheads) and fibers in a horizontal section through the
ventral part of the lateral pallium following an ipsilateral injection.
The star indicates the ventriculus lateralis posterior telencephali. B:
Labeled cell bodies (arrowheads) in the ipsilateral nucleus dorsalis
thalami. The asterisk indicates the fasciculus retroflexus. The arrow
indicates a labeled cell in the ventral thalamus. C: Retrogradely
labeled cell bodies in the contralateral nucleus ventralis thalami in
both periventricular (arrows) and laterally displaced (arrowhead)
positions. D: Labeled cells in the nucleus tuberculi posterioris (arrow)
and the nucleus ventralis hypothalami (arrowheads). Note that only
one labeled cell is located on the contralateral side of the nucleus
ventralis hypothalami (arrowhead on the right side). E: Labeled cells
of both cerebrospinal fluid- (CSF; arrows) and non-CSF-contacting
(arrowheads) types in the nucleus dorsalis and ventralis hypothalami.
F: Retrogradely labeled cells in the raphe region (arrows) following
BDA injection into the striatum. For abbreviations, see list. Scale
bars 5 200 µm in A,B,D,E, 100 µm in C,F.
STRIATAL AFFERENTS IN LAMPREY
77
Figure 3
78
Fig. 4. A–N: Schematic drawings of representative rostrocaudal
transverse sections showing the location of dopamine-immunoreactive
(DA-ir) perikarya (large dots, non-CSF-contacting cells; triangles,
CSF-contacting cells) and fibers (dashed lines and small dots). The
M.A. POMBAL ET AL.
sections are arranged from rostral to caudal, and the level of each
section is shown in the sagittal view of the brain in Figure 5A. For
abbreviations, see list. Scale bar 5 1 mm.
STRIATAL AFFERENTS IN LAMPREY
79
Figure 4
posterioris (ntp; Figs. 4G, 7F). These small (5–10 µm),
round cells were distributed around the midline, dorsal to
the recessus posterior. In another species of lamprey,
Petromyzon marinus, cells in this nucleus showed a stronger DA immunoreactivity and were somewhat larger and
more numerous (data not shown). This region has been
considered by Baumgarten (1972) as the cytoarchitectonic
area equivalent to the ventral tegmental area-substantia
nigra of other vertebrates.
Fibers. Dopaminergic cells in the preoptic region gave
rise to a diffuse fiber tract that coursed caudally towards
the hypothalamus, joining the tractus olfactohypothalamicus of Heier (1948), which passes by in close proximity
(Fig. 5B). This DA-ir fiber tract may innervate the neurohypophysis and, if so, form a well-developed neurosecretory
pathway (see also Nieuwenhuys, 1977). Some other fine
efferent fibers from the preoptic region followed and joined
the tractus opticus, also coursing caudally to thalamic
levels (Heier, 1948).
The mediobasal diencephalon displayed a very dense
plexus of DA-ir fibers, originating from CSF-contacting
cells of the dhy and the vhy (Figs. 5B, 7). After reaching the
lateral part of the brain, these immunopositive fibers gave
rise to two main fiber bundles. One coursed rostrally to the
telencephalon and innervated mainly the striatum region,
whereas the other projected caudally to the mesencephalon, the rhombencephalon, and even the dorsal column of
the rostralmost region of the spinal cord (Figs. 5B, 7C).
(Continued.)
Some labeled fibers also turned dorsally to innervate the
corpus geniculatum laterale (cgl; Fig. 4F) and the pretectal
region (Fig. 4G). A few DA-ir fibers were also seen crossing
in the commissura posterior (cp; Fig. 4G), continuing to the
contralateral pretectal region. In the epithalamus, the
habenula was almost completely devoid of DA-ir fibers
(Figs. 4E, 5B). DA-ir fibers were absent from the pineal
and parapineal organs (Figs. 4B,C, 5B). The neural lobe of
the hypophysis showed abundant DA-ir fibers, mainly in
its ventrocaudal aspect (Figs. 4D,E, 5B, 7C).
Mesencephalon
Perikarya. In the mesencephalon, only a few small (5
µm in diameter), round, weakly labeled DA-ir cells were
observed in the caudal part of the superficial stratum of
the tectum opticum (Figs. 4H, 5A, 8A). These cells were
monopolar, and their short basal processes coursed deeply
into the tectum opticum. No DA-ir cell bodies were detected in the ventral mesencephalon (Fig. 4H).
Fibers. The lamprey mesencephalon received a considerable dopaminergic innervation from the diencephalon.
Dorsally, the periventricular and central strata of the
tectum opticum received numerous DA-ir fibers (Figs. 4H,
5B, 8A), whereas the optic and superficial strata were
devoid of immunoreactive fibers. A moderate dopaminergic
innervation was present in the torus semicircularis, retinopetal nuclei (nucleus M5 of Schober and mesencephalic
80
M.A. POMBAL ET AL.
Fig. 5. Diagrams summarizing the distribution of DA-ir cells and
the main fiber tracts in the adult lamprey brain and spinal cord, as
projected in a sagittal view. A: Schematic drawing illustrating the
localization of DA-ir perikarya (dots). Levels A–N correspond to the
transverse sections shown in Figure 4. B: Main dopaminergic fiber
tracts (dashed lines) in a midsagittal view of the lamprey brain. Note
that the most prominent tracts, originating from the diencephalon,
course rostrally to the basal telencephalon and caudally to the
brainstem. Dots represent DA-ir perikarya as in A. For abbreviations,
see list. Scale bar 5 1 mm.
reticular area) and mesencephalic tegmentum (Fig. 4H).
Most of the caudally projecting dopaminergic fibers in the
mesencephalon were located in the region between the
oculomotor nucleus and the habenulointerpeduncular tract,
which, in itself, was devoid of immunopositive fibers along
its whole extent (Figs. 4G–I, 7F).
STRIATAL AFFERENTS IN LAMPREY
Rhombencephalon
Perikarya. Some weakly stained DA-ir cell bodies
were observed in the ventromedial part of the isthmic
nuclei (anterior rhombencephalic reticular nucleus or
nucleus isthmi rhombencephali ventralis medialis of Pierre
et al., 1992; Figs. 4I, 5A, 8B). These cells were round or
pear-shaped and had their basal processes directed toward
the lateral neuropil. In the rhombencephalon proper, a few
DA-ir cells were seen in the ventral nucleus of the octavolateral area, just dorsal to the most rostral part of the
descending trigeminal tract (Figs. 4J, 5A, 8C,D). These
cells were small (5–8 µm) and round or ovoid in shape.
Caudally, at the level of the posterior rhombencephalic
reticular nucleus, a row of small DA-ir cells was located
around the midline (Figs. 4L, 5A, 8E; CSF-contacting
type). Some DA-ir cells were also located dorsally near the
sulcus limitans of His in the presumed nucleus tractus
solitarii (Figs. 4L, 5A, 8E).
Fibers. The rhombencephalon was heterogeneously
innervated with DA-ir fibers, which were widely distributed along its rostrocaudal extent. In the isthmic region,
numerous DA-ir fibers were seen in the whole tegmentum
and in the so-called cerebellar commissura (Figs. 4I, 8B).
In the octavolateral area, dopaminergic fibers were abundant and clearly delineated the dorsal and medial nuclei
(Figs. 4J,K, 8C), where electro- and mechanoreceptive
fibers of the lateral line system are distributed (Ronan and
Northcutt, 1987). In addition, the ventral nucleus (Figs.
4J–L, 8C,D) of this area exhibited a dense and homogeneous innervation by DA-ir fibers. Most DA-ir fibers,
however, were distributed in the ventromedial tegmentum
of the basal plate. Some fibers also formed a diffuse
periventricular neuropil that bordered the motor nuclei
(V–XII; Figs. 4J–L, 8C–E) of the visceral column. In the
caudal rhombencephalon, most DA-ir fibers shifted dorsally to enter the dorsal column of the spinal cord (Figs.
4L,M, 8E,F).
81
Catecholaminergic innervation of the
lamprey striatum
BDA tracing was combined with indirect immunofluorescence for TH (n 5 8; see Materials and Methods) to study
the origin of the catecholaminergic innervation of the
lamprey striatum. The immunohistochemical results with
a polyclonal TH antibody were largely similar to those
reported by Pierre et al. (1994). Thus, we will describe only
briefly certain differences in order to compare TH- and
DA-ir structures (see also Table 1). In our experiments, no
TH-ir cells were found in the nucleus olfactorius anterior,
nucleus dorsalis thalami, nucleus commissurae posterioris, or nucleus tractus solitarii. Two different TH-ir cell
populations were present in the region of the posterior
tuberculum, i.e., the nucleus tuberculi posterioris, formed
by small, round, non-CSF-contacting cells located close to
the midline and the nucleus paratubercularis posterior,
which contains larger and lateroventrally displaced CSFcontacting cells.
Following BDA injections into the striatum combined
with TH immunocytochemistry, double-labeled cells were
found in both the nucleus tuberculi posterioris (Fig. 9A,B)
and the presumed locus coeruleus (Fig. 9C,D). In the
nucleus tuberculi posterioris, TH-ir cells abundant, with
strong immunoreactivity, and were somewhat clustered in
a small region (Fig. 9A). In the presumed locus coeruleus,
TH-ir cells were relatively few in number, with weak
immunoreactivity, and were scattered dorsoventrally in
the lateral part of the gray (Fig. 9C). The maximum
number of double-labeled cells was 7 of 16 retrogradely
labeled cells in the nucleus tuberculi posterioris and 5 of 8
retrogradely labeled cells in the locus coeruleus.
DISCUSSION
Spinal cord
The results of this study establish the presence of a
dense dopaminergic innervation of the neuropil of the
striatum in the lamprey. The origin of this innervation was
determined by combining TH immunohistochemistry with
tracing techniques (BDA) by means of double fluorescence.
Perikarya. Similar to the very caudal part of the
rhombencephalon, DA-ir cell bodies were found ventral to
the central canal along the whole extent of the spinal cord
(Figs. 4M,N, 5A, 8F). These cells were small (10 3 6 µm)
and bipolar and had a short apical process that reached the
central canal and terminated as a bulb in close apposition
to the Reissner fiber. Some non-CSF-contacting dopaminergic cells were also located ventral or slightly lateral to the
central canal (Figs. 4M,N, 8F). The detailed morphology
and synaptic connectivity of the intraspinal dopaminergic
system has been published previously (see Schotland et
al., 1996).
Fibers. Many descending DA-ir fibers from the brainstem were found in the dorsal part of the rostralmost
spinal cord (first segments; Figs. 4M, 8F). More caudally,
the number of these fibers decreased rapidly, and only a
few could be followed to the trunk region. Some DA-ir
fibers originated from CSF-contacting cells and formed a
small dopaminergic plexus dorsal to the central canal,
whereas other coursed to the lateral gray matter (Schotland et al., 1996). Ventrally directed processes from intrinsic non-CSF-contacting dopaminergic cells gave rise to a
prominent fiber plexus in the ventromedial column of the
spinal cord (Figs. 4M,N, 8F; Schotland et al., 1996).
Injections into the striatum region were performed
through the telencephalic ventricle but not from the
external part of the brain, in order to avoid or minimize the
labeling of ascending and descending fibers that course in
the lateral forebrain bundle and tractus opticus. In some
experiments, however, parts of these fiber tracts were also
labeled.
After injections into the striatum, numerous cell populations from a range of telencephalic, diencephalic, and
rhombencephalic sources were identified, some of which
have been reported previously (Polenova and Vesselkin,
1993; Northcutt and Wicht, 1997; see below). The numbers
of afferent striatal cells in the nucleus tuberculi posterioris
that project to the striatum were comparatively low, even
after a long transport time. In some experiments with
FDA, no labeled cells were obtained in the caudal diencephalon (dorsally in the nucleus dorsalis thalami and
ventrally in the nucleus tuberculi posterioris). In immunohistochemical experiments, several cells were stained for
DA in the nucleus tuberculi posterioris; thus, it could be
anticipated that this nucleus has more prominent projections to striatum. Retrogradely labeled cells in the pre-
Technical comments
82
M.A. POMBAL ET AL.
Figure 6
STRIATAL AFFERENTS IN LAMPREY
sumed locus coeruleus and the raphe region were obtained
only when BDA was used as a tracer. Although similar
survival times allow in vitro labeling of the striatal
afferents, no labeled cells were seen in the nucleus of the
solitary tract of the lamprey. The low number of retrogradely labeled cells in some striatal sources and the
absence of labeled cells in the nucleus of the solitary tract
could be explained by the long transport distance between
these structures and the striatum and the small diameter
of the retrogradely labeled fibers. The limitations of the
technique for retrograde labeling, thus, should be taken
into account when interpreting negative results.
Striatal afferents from the dorsal thalamus
Based on the number of retrogradely labeled cells, the
major input to the striatum was the nucleus dorsalis
thalami. In stained histological material, fibers apparently
originating from the striatal area were traced to the
nucleus dorsalis thalami by Johnston (1902) and Tretjakoff (1909), forming the tractus thalamofrontalis. Injections of DiI in the dorsal thalamus (Ichthyomyzon unicuspis; Northcutt and Wicht, 1997) also revealed the presence
of varicose fibers in the neuropil lateral to the ipsilateral
striatum. However, such a projection was not revealed by
injection of HRP and HRP-wheat germ agglutinin (WGA)
(L. fluviatilis; Polenova and Vesselkin, 1993). Projections
from the nucleus dorsalis thalami to the striatum have
been shown in amphibians (Wilczynski and Northcutt,
1983; Wicht and Himstedt, 1988; Dubé et al., 1990; González et al., 1994c; Marı́n et al., 1997a) and reptiles (Parent,
1976; Lohman and van Woerden-Verkley, 1978; González
et al., 1990). The thalamic intralaminar nucleus in mammals sends a prominent projection to the striatum, whereas
a minor input is provided by the lateral posterior/pulvinar
complex (see Butler, 1994).
The lamprey nucleus dorsalis thalami receives direct
connections from the contralateral retina (Vesselkin et al.,
1980; De Miguel et al., 1990), all major divisions of the
pallium, the nucleus praeopticus, hypothalamus, optic
Fig. 6. DA-ir cells and fibers in the forebrain of the river lamprey.
A: Sagittal section through the evaginated part of the lamprey
telencephalon showing DA-ir cells in the internal granular layer (igl)
of the olfactory bulb. Note also the presence of immunoreactive
terminals in the dorsomedial telencephalic neuropil (dm) as well as
immunoreactive fibers in the nonolfactory part of the telencephalon.
The dashed line shows the limit between the olfactory (right) and
nonolfactory (left) parts of the telencephalon. B: Sagittal section
illustrating DA-ir cell bodies in the nucleus commissurae anterior
(nca), nucleus praeopticus (npo), and nucleus commissurae postopticae (ncpo). The arrow points to a displaced cell of the nucleus
praeopticus. The star indicates the recessus praeopticus. C: Transverse section showing DA-ir cells in the nucleus praeopticus (npo) and
DA-ir fibers in the basal telencephalon, which are particularly abundant in the periventricular neuropil that borders the striatum (st).
Note that some of the non-CSF-contacting DA-ir cells are dorsally
displaced (arrows). D: Horizontal section showing the dopaminergic
innervation of the basal telencephalon. A conspicuous dopaminergic
plexus is located medial to the striatum (st), whereas only a few fibers
course to the lateral evaginated part (star). E: Horizontal section
showing CSF-contacting cells in the nucleus praeopticus (npo) and
nucleus dorsalis hypothalami (dhy) and non-CSF-contacting cells in
the nucleus commissurae postopticae (ncpo). The star indicates the
recessus praeopticus. A dense plexus of dopaminergic fibers is seen in
the basal plate, whereas the tractus opticus (to) is devoid of immunoreactive fibers. For other abbreviations, see list. Scale bars 5 200 µm in
A–C,E, 400 µm in D.
83
tectum, midbrain tegmentum, and dorsal isthmal gray as
well as secondary and tertiary olfactory projections (Polenova and Vesselkin, 1993; Northcutt and Wicht, 1997). It
has also been suggested that thalamic nuclei receive
projections from the spinal cord (spinothalamic tract),
cerebellum, area octavolateralis, and parietal organs (Heier,
1948; Nieuwenhuys, 1977). Thus, it appears that sensory
information from many different sources is processed in
the nucleus dorsalis thalami and then relayed to coordinating telencephalic centers, such as the striatum.
Cells of the nucleus ventralis thalami are also retrogradely labeled after injections of FDA into the striatum,
suggesting that they also project to this nucleus. Projections from the nucleus ventralis thalami appear to arise
bilaterally, with the fibers crossing in the commissura
postopticae. A small number of retrogradely labeled cells
has recently been reported in the ventral thalamus of both
anuran and urodele amphibians (see Marı́n et al., 1997a).
Striatal dopaminergic afferents
Previous results show that the striatum in lamprey
receives a strong catecholaminergic innervation (Brodin et
al., 1990b; Pierre et al., 1994). Here, we report that this
innervation is mainly dopaminergic. In most vertebrates,
this dopaminergic innervation originates mainly from the
ventral mesencephalon. Because no catecholaminergic cells
were found in the lamprey mesencephalon, those located
rostrally to the dimesencephalic boundary in the posterior
tubercle were supposed to correspond to the ventral tegmental area or substantia nigra of other vertebrates
(Baumgarten, 1972). In the present study, DA-ir cells were
found in the nucleus tuberculi posterioris, whereas no
DA-ir cells were detected in the nucleus paratubercularis
posterior, both of which contain TH-ir cells (Brodin et al.,
1990b; Pierre et al., 1994; present results). The combination of retrograde tracing techniques (BDA) with TH
immunohistochemistry demonstrated that some cells in
the nucleus tuberculi posterioris that project to the striatum are dopaminergic. Catecholaminergic cells in the
region of the posterior tubercle have also been identified in
amphibians (Bartels, 1971; Wilczynsky and Northcutt,
1983; Wicht and Himstedt, 1988; González and Smeets,
1991; González et al., 1993, 1994a–c). This region, together
with the medial part of the mesencephalic tegmentum,
provides the dopaminergic innervation of the amphibian
striatum (González et al., 1994c; Marı́n et al., 1997b). The
catecholaminergic cells of the posterior tubercle in lamprey were regarded by Baumgarten (1972) as part of a
structure that migrates into the base of the midbrain in
tetrapods. Such a migration of cells has not yet been
clearly demonstrated. The rostrocaudal gradient in the
time of appearance of the posterior tubercle/midbrain
TH-ir/DA-ir cells in amphibians during development favors this hypothesis (González et al., 1994a,b, 1995). This
developmental pattern of a single merged cell group is also
consistent with the embryonic origin of the substantia
nigra (A9) and the ventral tegmental (A10) nuclei in
mammals (see Fallon and Loughlin, 1985).
The caudal portion of the striatum is less densely
innervated by dopaminergic axons. Baumgarten (1972),
however, described a few small, fluorescent, bipolar CSFcontacting cells with weakly fluorescent axons arborizing
locally within the subependymal fiber layer. By using the
streptavidin-biotin procedure, we also found a few weakly
84
M.A. POMBAL ET AL.
Figure 7
STRIATAL AFFERENTS IN LAMPREY
stained DA-ir cells in this region that appeared to make
contact with the surface of the ventricle.
Striatal afferents from the hypothalamus
The injections of FDA into the striatum revealed large
numbers of retrogradely labeled cells in both the ipsi- and
contralateral hypothalamus. Considerable numbers of cells
of both CSF and non-CSF-contacting types were labeled in
the ipsilateral hypothalamus, whereas only occasional
cells were labeled on the contralateral side. Part of the
dorsal hypothalamus where 5-HT (Brodin et al., 1990b;
Pierre et al., 1992), TH (Pierre et al., 1994; present
results), and DA (present results) immunoreactivities are
found may correspond to the paraventricular organ (PVO)
of gnathostomes (Pierre et al., 1994; Pombal and Puelles,
unpublished results). It has been suggested that the PVO
of the hypothalamus of amphibians and reptiles may
provide a massive monoaminergic innervation of the striatum (Sims, 1977; Tohyama et al., 1977; Dubé and Parent,
1982; Wilczynski and Northcutt, 1983; Dubé et al., 1990).
However, recent studies with double-labeling combining
tract-tracing and immunohistochemistry for DA or TH
have demonstrated that the PVO does not project to the
striatum in either anurans or urodeles (González et al.,
1994c; Marı́n et al., 1997b). The same experiments also
revealed striatal afferent cells in the ventral hypothalamus in these two species (González et al., 1994c; Marı́n et
al., 1997a). Our results show that nondopaminergic hypothalamic structures also project to the striatum in lampreys.
Striatal afferents from the olfactory bulb,
preoptic region, and pallium
Some retrogradely labeled cells were present medially in
the olfactory bulb after injection of FDA into the striatum.
The presence of olfactory bulb afferents to the striatum is
in agreement with previous reports of labeled axonal
terminals in the striatum after HRP or HRP-WGA injections into the olfactory bulb (Northcutt and Puzdrowski,
1988; Polenova and Vesselkin, 1993). Striatal projections
from the olfactory bulb (Northcutt and Royce, 1975; Dubé
Fig. 7. Photomicrographs of examples of DA-ir cells and fibers in
the mediobasal diencephalon of the river lamprey. A,B: Transverse
sections showing numerous DA-ir CSF-contacting cell bodies in the
nucleus dorsalis hypothalami. Most labeled fibers course lateroventrally and then turn rostrally or caudally. C: Sagittal section illustrating the dopaminergic innervation in diencephalon (D), mesencephalon
(M), and rostral rhombencephalon (R). DA-ir fibers, mainly originating from the ventral diencephalon, course caudally to innervate the
brainstem. Note also the strong dopaminergic innervation of the
neurohypophysis (arrow). D: Sagittal section through the ventrocaudal hypothalamus showing numerous CSF-contacting DA-ir cells in
the nucleus commissurae postinfundibularis and nucleus ventralis
hypothalami (arrow). Note also that not all cell bodies surrounding the
recessus posterior (star) are immunopositive. A dense dopaminergic
plexus appears caudally to this recessus. E: Transverse section
showing numerous CSF-contacting cells in the nucleus commissurae
postinfundibularis, surrounding the recessus posterior (rpos). F: Horizontal section showing CSF-contacting DA-ir cells in the nucleus
ventralis hypothalami (vhy) and non-CSF-contacting cells in the
nucleus tuberculi posterioris (ntp). Note also the presence of a dense
dopaminergic lateral plexus, whereas the decussation of the fasciculus
retroflexus (f) is free of labeled fibers. For other abbreviations, see list.
Scale bars 5 400 µm in A,B, 500 µm in C, 200 µm in D–F.
85
et al., 1990; González et al., 1994c; Marı́n et al., 1997a)
have also been reported in amphibians.
The preoptic region provides afferents to the striatum.
The preoptic area has also been shown to be reciprocally
connected with the striatum via the medial forebrain
bundle in amphibians (Wilczynski and Northcutt, 1983;
Dubé et al., 1990; Marı́n et al., 1997a). Although this
region, together with the suprachiasmatic nucleus, was
suggested as another possible source of the dopaminergic
innervation of the amphibian striatum, recent doublelabeling experiments did not support this hypothesis
(González et al., 1994c; Marı́n et al., 1997b).
Large numbers of cell bodies were retrogradely labeled
in both the dorsal and the ventral parts of the ipsilateral
lateral pallium after injections into the striatum, and a few
were found in the ipsilateral, medial, and dorsal pallium,
suggesting that all major pallial divisions project to this
nucleus. Injections of HRP, HRP-WGA, or DiI in the lateral
and medial pallium also demonstrated efferents to the
striatum (Polenova and Vesselkin, 1993; Northcutt and
Wicht, 1997). Although the latter authors described terminals from the medial pallium bilaterally in the striatum,
only ipsilateral efferents were observed in the present
study. Retrogradely labeled cells in the medial pallium of
amphibians (González et al., 1994c; Marı́n et al., 1997a)
and the lateral pallium of Caiman crocodilus (Brauth and
Kitt, 1980) have also been described after injections of
different tracers into the striatum.
Striatal afferents from the rhombencephalon
In the present study, some retrogradely labeled cells
were found in the ipsilateral isthmus region, in the
presumed locus coeruleus, and also in the raphe region,
just ventral to the trigeminal motor nucleus and close to
the midline. In amphibians, a few cells located in the
midbrain tegmentum, locus coeruleus, parabrachial
nucleus, raphe nuclei, different reticular nuclei, and the
nucleus tractus solitarii give rise to ascending fibers that
reach the striatum region (González et al., 1994c; Smeets
et al., 1995; Marı́n et al., 1996, 1997a,b). In reptiles, cells of
the nucleus tegmenti pedunculopontinus, locus coeruleus,
and the raphe system also project to the ventrolateral area
of the telencephalon, which is a presumed homologue of
the mammalian striatum (Brauth and Kitt, 1980). In
birds, some neurons located near the midline of the caudal
midbrain tegmentum (Wild, 1987) and within the locus
coeruleus, nucleus subcoeruleus dorsalis, and nucleus
subcoeruleus ventralis (Kitt and Brauth, 1986) project to
the paleostriatum. In mammals, the dorsal raphe nucleus
(Steinbusch et al., 1981a) and probably also the locus
coeruleus (Parent et al., 1983) provide inputs to the
striatum.
Some double-labeled cells were found in the isthmic
region after BDA application in the striatum in combination with TH immunohistochemistry, which, thus, can be
considered homologous to the locus coeruleus of amniotes.
Similar results have also recently been reported in amphibians (Marı́n et al., 1996, 1997b).
No structures caudal to the raphe region were found to
project to the lamprey striatum. Even in our best experiment, only a few cells were retrogradely labeled in the
raphe region after 65 hours of tracer transport in vitro (see
above). Thus, we cannot rule out the existence in lamprey
of other caudally located structures projecting to the
striatum, like the nucleus tractus solitarii, where some
Figure 8
STRIATAL AFFERENTS IN LAMPREY
87
TABLE 1. Comparative Analysis of the Catecholaminergic System in the Lamprey Brain and Spinal Cord and of the Projections to the Striatum1
T
D
M
R
SC
Cell
populations
igl
noa
nca
npo
ncpo
dth
hy
ncpi
ntp2
ncp
ot
arn3
vn
nts
cc
Vcc
Lcc
TH-ir (1)
TH-ir (2)
DA-ir (2)
DA-ir (3)
st (4)
1
1
1
2
1
1
2
2
2
2
2
2
1
2
1
1
1
1
2
1
1
1
1
2
1
1
2
2
2
1
1
1
1
1
1
1
1
1
1
2
1
1
1
2
1
1
1
6
2
2
2
2
1
2
2
1
1
1
2
1
2
2
1
2
2
1
2
1
2
0
1
1
1
2
2
2
2
1
2
2
2
2
1
2
2
1The
presence (1) or absence (2) of immunoreactivity (-ir) or inputs is indicated in the different regions obtained from the results of 1) Pierre et al. (1994; adult river lamprey), 2)
present study, 3) Yáñez et al. (1992; larval sea lamprey), and 4) present study.
different cell populations were described in ntp, both were immunoreactive to tyraxine hydroxylase (TH) but only one to dopamine (DA; see Discussion).
3Includes both nucleus isthmi rhombencephali dorsalis posteior and nucleus isthmi rhombencephali ventralis medialis (which includes the presumed locus coeruleus) of Pierre et al.
(1994). 6, Only weakly dopamine (DA) ir cells were detected by using streptavidin-biotin (SAB) complex; 0, no cells were retrogradely labeled in this nucleus following injection of
fluorescein-coupled dextran-amine (FDA) or BDA in striatum. Lcc, lateral to the central canal; Vcc, ventral to the central canal. The different regions are defined in the list of
abbreviations.
2Two
DA-ir cells were also located (present results), or reticular
nuclei.
Dopaminergic system in cyclostomes
The general distribution pattern of DA-ir neurons in the
adult river lamprey brain, in general, is similar to that of
TH-ir cells reported in the same species (Pierre et al.,
1994), but several differences deserve a comment (see
Table 1). 1) Some cell populations (the nucleus olfactorius
anterior, nucleus dorsalis thalami, pretectum, and nucleus
isthmirhombencephali dorsalis posterior) were positive for
TH (Pierre et al., 1994) but not for DA or TH (present
study). This difference might be related to the use of
different TH antibodies. Another possibility is that L-3,4Dihydroxyphenylalanine (L-DOPA) could be the biologi-
Fig. 8. Dopamine-immunoreactive cells and fibers in the brainstem and rostralmost spinal cord of the river lamprey. A: Transverse
section through the lamprey tectum opticum showing some small
dopaminergic cell bodies (arrows) in the superficial stratum (ss). Note
also the presence of DA-ir fibers in the periventricular (ps) and the
central strata (cs). B: Transverse section through the isthmic region
showing some weakly DA-ir cell bodies (arrows) in the ventromedial
part of the isthmic nuclei as well as numerous immunopositive fibers.
C: Transverse section at the level of the nucleus motorius nervi
trigemini showing the dopaminergic innervation at this level. Numerous immunopositive fibers are distributed in the ventromedial rhombencephalic tegmentum, whereas the region of trigeminal motoneurons
(Vm) is devoid of labeled fibers. In the octavolateral area, both dorsal
(dn) and medial (mn) nuclei are clearly delineated by dopaminergic
fibers, whereas some fibers are located in the ventral nucleus of this
area, where two small dopaminergic cell bodies (small arrows) are also
present. A few labeled fibers are also intermingled within the radix
sensitivus nervi trigemini (dV). D: Magnification of a parallel section
showing a dopaminergic cell (arrow) in the ventral nucleus of the
octavolateral area (vn), just over the rostralmost part of the radix
sensitivus nervi trigemini (dV). Note also the presence of dopaminergic fibers in the radix sensitivus nervi trigemini (dV). E: Transverse
section through the caudal rhombencephalon showing small CSFcontacting DA-ir cells (small straight arrows) in the midline (sulcus
medianus) and slightly displaced as well as one DA-ir cell in the
presumed nucleus tractus solitarii (large straight arrow). Note also
the rich dopaminergic ventromedial plexus originating from the small
CSF-contacting cells as well as the pattern of innervation in the
rhombencephalic tegmentum. The curved arrow indicates one root of
the nervi vagi. F: Transverse section of the rostralmost spinal cord
showing DA-ir cells contacting the central canal as well as some
non-CSF-contacting cells (small solid arrows) displaced ventrally or
laterally. Numerous dopaminergic fibers descending from the brain
distributed in the dorsal and dorsolateral parts of the spinal cord. A
dense dopaminergic plexus originating from intrinsic dopaminergic
cells is evident in the ventromedial part of the section. The large open
arrow points to a spinal ventral root. For other abbreviations, see list.
Scale bars 5 100 µm for A,B,D,E, 300 µm in C, 400 µm in F.
cally active catecholamine, as suggested in some cell
populations in the mammalian hypothalamus (Okamura
et al., 1988; Vincent and Hope, 1990; Mons et al., 1991), in
reptiles (Smeets and Steinbusch, 1990) and in amphibians
(González and Smeets, 1994).
2) Other cell populations (the nucleus commissurae
anterior, superficial stratum of the tectum opticum, ventral nucleus of the octavolateral area, and some non-CSFcontacting cells located ventral and lateral to the spinal
central canal) were positive for DA but not for TH. It is
possible that the low level of TH in these small cell
populations cannot be detected with these techniques or
that these are DA-accumulating cells that do not synthesize this amine, as pointed out by Smeets and González
(1990). Similar discrepancies between the distribution of
DA-containing cells and TH-ir cells have been observed in
different cell populations of other vertebrates (for review,
see Manso et al., 1993).
3) In the region of the posterior tubercule, the distribution and size of DA-ir and TH-ir (Brodin et al., 1990b;
Pierre et al., 1994; present results) appears to be different.
The small cells located in the nucleus tuberculi posterioris
were both DA-ir and TH-ir, whereas the larger cells in the
nucleus paratubercularis posterior were only TH-ir.
There are two recent studies on the catecholaminergic
innervation of the hagfish brain. Presumed catecholaminergic cells were found in the preoptic region, the hypothalamus (CSF- and non-CSF-contacting types), and the isthmal region of Eptatretus stouti by using a TH antibody
(Wicht and Northcutt, 1994). DA-ir cells of both CSF- and
non-CSF-contacting types were found immunohistochemically only in the hypothalamus of Eptatretus burgeri
(Kadota et al., 1993). Curiously enough, these results are
in agreement with the distribution of DA-ir cells in the
larval lamprey, where most immunopositive cell bodies are
located in the hypothalamus and have apical dendrites
that reach the surface of the ventricle (Table 1; Yáñez et al.,
1992). Although DA1 fibers are widely distributed throughout the brain and spinal cord, it appears that the dopaminergic system in hagfishes is much more restricted than in
adult lampreys (present results).
Comparison with other vertebrates
A recent review of the phylogenetic and developmental
aspects of the catecholaminergic systems in the vertebrate
central nervous system (Smeets and Reiner, 1994b) revealed that most catecholaminergic cell groups are present
in all species studied so far. The present results demon-
88
M.A. POMBAL ET AL.
Fig. 9. Photomicrographs of double-labeled cells following BDA
injection into the left striatum combined with indirect immunofluorescence for tyrosine hydroxylase (TH). A,B: Horizontal section through
the nucleus tuberculi posterioris showing two double-labeled cells
(arrows in A, TH-ir cells; arrows in B, BDA retrogradely labeled cells).
C,D: Transverse section through the isthmic region showing two
double-labeled cells in the presumed nucleus coeruleus (arrows in C,
TH-ir cells; arrows in D, BDA retrogradely labeled cells). Scale bar 5
100 µm.
strate that the lamprey brain also has a well-developed
dopaminergic system.
In addition to this general scheme, some groups of
catecholaminergic cells were found in the present study
but were not observed previously: 1) A limited number of
small DA-ir cells are present in the superficial stratum of
the tectum opticum (present results). Small TH-ir cells
were also identified in the most superficial and rostral
intermediate layers of the tectum opticum in Heterodontus and Squalus, respectively (Stuesse et al., 1994).
2) Some small DA-ir cells are also present in the
boundary between the ventral nucleus of the octavolateral
area and the descending trigeminal tract. Catecholaminergic cells have been described in a similar position in
Apteronotus (see Meek, 1994), which was referred to as
nucleus of the descending trigeminal tract.
3) A CSF-contacting DA-ir cell group is present in
the midline of the caudal lamprey brainstem. DA-ir
ependymal and subependymal cells were also found in
the same location in Rana and Pleurodeles by González
and Smeets (1991). This dopaminergic cell population
was thought to represent a derived character of amphibians, because it had not been observed in any other
vertebrate.
3) In the lamprey spinal cord, in addition to CSFcontacting cells described in the spinal cord of most
vertebrates (Pierre et al., 1994; Smeets and Reiner, 1994b;
Schotland et al., 1995; present results), some non-CSFcontacting, DA-ir cells are also present ventrally and
slightly lateral to the central canal (Schotland et al., 1995;
present results). It has also been demonstrated that these
cells colocalize 5-hydroxytryptamine (5-HT; Schotland et
al., 1995; Zhang et al., 1996), which has additive effects in
their action as modulators of spinal neurons controlling
locomotion (Schotland et al., 1995).
STRIATAL AFFERENTS IN LAMPREY
Other features of the dopaminergic system
in the river lamprey
The particular location of the CSF-contacting DA-ir cells
in the preoptic region, overlying the chiasma opticum,
clearly resembles that of the dopaminergic suprachiasmatic cell population of other vertebrates (for review, see
Smeets and Reiner, 1994b). This dopaminergic cell group,
labeled here as the nucleus commissurae anterior, also
contains CSF-contacting cells in the recessus praeopticus.
Thus, it could be the homologue of the anterior preoptic
region of other vertebrates.
An important feature is the high number of DA-ir
CSF-contacting cell bodies in the hypothalamus of lampreys compared with other vertebrates. This finding, together with observations made in amphibians (González
and Smeets, 1994) and reptiles (Smeets, 1994), supports
the notion that there is a reduction of CSF-contacting
catecholaminergic cells from fishes to mammals (Parent et
al., 1984).
The mesencephalic dopaminergic cell group, including
the retrorubral (A8), substantia nigra (A9), and ventral
tegmental area (A10), has not been observed in cyclostomes (Kadota et al., 1993; Pierre et al., 1994; present
results), holocephalians, and actinopterygian fish (Smeets
and Reiner, 1994b). A correlation throughout phylogeny
between a decreased PVO in the diencephalon and an
increase in the number and distribution of monoamine
neurons within the mesencephalon was suggested (Parent
et al., 1984). Although the available data in lampreys
support this correlation, recent data showing numerous
TH-ir cell bodies in the hypothalamus of both anamniotes
and amniotes, together with well-developed dopaminergic
cell groups in the midbrain (Smeets and Reiner, 1994b), do
not appear to support such a notion.
CONCLUSIONS
The striatum in lamprey receives inputs from different
regions of the brain and a dense dopaminergic innervation
that originates from the nucleus tuberculi posterioris in
the diencephalon. The overall dopaminergic innervation of
the lamprey brain shows several striking similarities with
the general vertebrate scheme; however, it also presents
some differences. The largest number of DA-ir cells occurs
in the hypothalamus, which is a common feature of the
dopamine system in fishes and amphibians, whereas no
DA-ir cells were found in the midbrain tegmentum, which
represents the most prominent catecholaminergic population of amniotes.
ACKNOWLEDGMENTS
We thank Drs. H.W.M. Steinbusch and T. Hökfelt for
generous supply of DA and TH antisera and Drs. L.
Brodin, D. Parker, and P. Wallén for helpful comments on
the paper. We also thank H. Axegren, M. Bredmyr, and M.
Podal for skilful technical assistance.
This work was supported by the Swedish Medical Research Council (project 3026 and 11562) and by fellowships
from the Xunta de Galicia (Spain), the European Science
Foundation, and the University of Vigo to M.A.P.
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combined, immunohistochemical, dopaminergic, afferents, stud, tracing, striatum, references, lamprey, system, special
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