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Connections of the basal forebrain of the weakly electric fish Eigenmannia virescens

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THE JOURNAL OF COMPARATIVE NEUROLOGY 389:49–64 (1997)
Connections of the Basal Forebrain
of the Weakly Electric Fish,
Eigenmannia virescens
CALVIN J.H. WONG*
The Neurobiology Unit, Scripps Institution of Oceanography,
University of California San Diego, La Jolla, California 92093-0201
ABSTRACT
The organization of the ventral nucleus of the ventral telencephalon (Vv) was examined in
the weakly electric fish, Eigenmannia virescens. This nucleus, which is considered the teleost
homologue to the basal forebrain nuclei of other vertebrates, was subdivided into dorsal and
ventral subdivisions, based upon cytoarchitectonic, immunohistochemical, and connectional
criteria. Afferent projections were observed from the medial olfactory bulb as well as the
terminal nerve ganglion. Telencephalic afferents to the Vv were very restricted, consisting of
the supracommissural and the dorsal intermediate nuclei of the ventral telencephalon, the
nucleus taenia, and the medial region of the posterior nucleus of the dorsal telencephalon.
However, the major afferents to the Vv were diencephalic, particularly those originating from
the rostral preoptic area and other hypothalamic nuclei. Additional afferents included the
posterior tubercular nucleus, the locus coeruleus, the medial perilemniscal nucleus, and the
periventricular nucleus of the posterior tuberculum. Relatively weak projections were
observed from the ventral thalamus and the dorsal posterior thalamic nucleus. As described
previously, the diencephalic complex of the central posterior thalamic nucleus/prepacemaker
nucleus (CP/PPn), which also has cells that innervate the pacemaker circuitry controlling the
production of an electric organ discharge, projects to the Vv.
Terminal fields of the Vv were observed to be coextensive with afferent cell groups in the
preoptic area, lateral and caudal hypothalamic nuclei, and thalamus. An additional efferent
target of the Vv was the pretectal nucleus electrosensorius. That many cell groups that are
connected with the Vv are also connected with the CP/PPn, particularly the preoptic and
hypothalamic nuclei, suggests that the electrocommunicatory system is intimately linked with
basal forebrain limbic pathways. J. Comp. Neurol. 389:49–64, 1997. r 1997 Wiley-Liss, Inc.
Indexing terms: septum; hypothalamus; reproduction; medial forebrain bundle; teleost
The basal forebrain and preoptic nuclei of teleost fishes
have been regions of considerable interest due to their role
in neuroendocrine control (Peter and Fryer, 1983) and the
motivation of reproductive and aggressive behaviors (Demski and Knigge, 1971; see Demski, 1983, for a review;
Satou et al., 1984; Fine and Perini, 1994), displaying many
functional similarities to homologous regions in other
vertebrates. Most anatomical studies on these regions in
teleosts have focused on connections with the olfactory
bulb (e.g., Bass, 1981b, 1981c; Levine and Dethier, 1985;
Sas et al., 1993; Matz, 1995) or projections to the pituitary
(Peter and Fryer, 1983; Johnston and Maler, 1992), whereas
examination of other connections with these areas has
been impaired, in part, due to their deep location and their
proximity to the forebrain bundle. Previous experimental
studies on the ventral and supracommissural nuclei of the
ventral telencephalon (Vv/Vs; Shiga et al., 1985a, 1985b;
r 1997 WILEY-LISS, INC.
Sloan, 1989) have identified connections with the preoptic
area, hypothalamus, and thalamus. However, as injections
or lesions were not necessarily confined to a single nucleus,
the specific connectivity of individual nuclei remains unknown.
Recent tract-tracing studies in the weakly electric fish,
E. virescens, identified reciprocal connections between the
electrocommunicatory system and the ventral telencephalon, the preoptic area, as well as more caudal regions of the
Grant sponsor: NIMH; Grant number: R37 MH26149-18; Grant sponsor:
NINCDS; Grant number: RO1 NS22244-08; Grant sponsor: NSF; Grant
number: BNS-9106705.
*Correspondence to: Dr. Calvin J.H. Wong, Department of Biological
Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9 Canada.
Received 11 February 1997; Revised 23 June 1997; Accepted 1 July 1997
50
C.J.H. WONG
hypothalamus (Keller et al., 1990; Heiligenberg et al.,
1991; Wong, 1997a). These findings corroborate previous
immunohistochemical studies that identified peptidergic
and monoaminergic innervation of diencephalic electrosensory/motor regions (see Zupanc and Maler, 1997, for a
review).
Gymnotiform fish produce an electric field, in the water
around them, called the electric organ discharge (EOD; see
Bass, 1986, for a review), of which they can modify the
temporal and spectral characteristics for social communication (Hopkins, 1974; Hagedorn and Heiligenberg, 1985;
Hopkins, 1988). Two diencephalic regions that act as a
sensory-motor interface have been identified to be involved
in the detection and production of electrocommunicatory
signals. One is the pretectal complex of the nucleus
electrosensorius (nE), which receives ascending input from
the electrosensory portion of the torus semicircularis (Carr
et al., 1981; Keller et al., 1990). Another region is the
thalamic complex of the central posterior nucleus/ prepacemaker nucleus (CP/PPn; Zupanc and Zupanc, 1992; Zupanc
and Heiligenberg, 1992), which receives input from the nE
(Keller et al., 1990; Heiligenberg et al., 1991; Wong,
1997a). The CP/PPn then, in turn, projects to the medullary pacemaker nucleus (Heiligenberg et al., 1981; Kawasaki et al., 1988; Stroh and Zupanc, 1995; Heiligenberg
et al., 1996; Wong, 1997a), which sets the timing of the
EOD (Dye and Meyer, 1986). Stimulation of the CP/PPn
can elicit modulations in the EOD, mimicking those that
occur naturally (Kawasaki and Heiligenberg, 1988; Ka-
wasaki et al., 1988), suggesting that other regions that
project to the CP/PPn might also influence electrocommunicatory behavior. In an effort to expand our understanding of forebrain pathways involved in electrocommunication in gymnotiform fish, the organization and connections
of one of the major telencephalic regions connected with
the CP/PPn, the Vv, were examined. Some results from
these experiments, regarding connections with the CP/
PPn, have been published previously (Wong, 1997a).
MATERIALS AND METHODS
Adult specimens of both sexes of Eigenmannia virescens
were used for experiments (length 10–20 cm). Fish were
obtained from a commercial supplier (Bailey’s Tropical
Fish, San Diego, CA) and maintained in aquaria containing water of 20 to 30 kOhms/cm resistivity and neutral pH,
at a temperature of 26–28°C. Fish were kept on a diurnal
cycle of 12 hours light/12 hours dark and fed live blackworms ad libitum. Animal care, anesthesia, surgery, and
killing were carried out in compliance with guidelines set
out by the Animal Subjects Committee for the University
of California, San Diego.
Normal anatomy
The normal anatomy of the telencephalon was examined
from the reference series of E. virescens, from the library of
the Heiligenberg laboratory. These series were transverse
Abbreviations
AC
ATh
cG
CE
CP/PPn
CR-l-ir
Dc
Dd
DFl
Dl
Dld
Dlp
Dlv
Dm1
Dm2
Dm2v
Dp
Dpm
DPn
Ec
EOD
Er
TS
FB
G
Ha
Hc
Hd
Hl
Hv
ICL
JAR
LCe
LFB
LL
MFB
MgT
MOB
nAPv
anterior commissure
anterior thalamic nucleus
commissure of Goldstein
central nucleus of the inferior lobe
complex of the central posterior nucleus (thalamus)
and the prepacemaker nucleus
calretinin-like immunoreactivity
central division of the dorsal telencephalon
dorsal division of the dorsal telencephalon
lateral diffuse nucleus of the inferior lobe
dorsolateral telencephalon
dorsolateral telencephalon, dorsal subdivision
dorsolateral telencephalon, posterior subdivision
dorsolateral telencephalon, ventral subdivision
dorsomedial telencephalon, subdivision 1
dorsomedial telencephalon, subdivision 2
dorsomedial telencephalon, subdivision 2, ventral
dorsoposterior telencephalon
dorsoposterior telencephalon, medial subdivision
dorsal posterior nucleus (thalamus)
caudal entopeduncular nucleus
electric organ discharge
rostral entopeduncular nucleuse
torus semicircularis efferents
forebrain bundle
glomerular nucleus
anterior hypothalamic nucleus
caudal hypothalamic nucleus
dorsal hypothalamic nucleus
lateral hypothalamic nucleus
ventral hypothalamic nucleus
internal cellular layer
jamming avoidance response
locus coeruleus
lateral forebrain bundle
lateral lemniscus
medial forebrain bundle
magnocellular tegmental nucleus
medial olfactory bulb
anterior periventricular nucleus
nE
nE<
nLTa
nPPv
nRLl
nT
OB
PC
PeG
PGl
PGm
PGr
PLm
POC
PPa
PPn
PPp
R
Rd
SPPn
TA
tBH
TeO
TP
TPP
TSd
TSv
Vc
Vd
Vi
Vid
Vir
Vl
VMTh
Vp
Vs
Vv
Vv-d
Vv-v
*
nucleus electrosensorius
nucleus electrosensorius, ‘down’subdivision
anterior region of the lateral tuberal nucleus
posterior periventricular nucleus
lateral nucleus of the lateral recess
nucleus taenia
olfactory bulb
posterior commissure
periglomerular nucleus
preglomerular complex, lateral subdivision
preglomerular complex, medial subdivision
preglomerular complex, rostral subdivision
medial perilemniscal nucleus
postoptic commissure
anterior periventricular preoptic nucleus
prepacemaker nucleus
posterior periventricular preoptic nucleus
red nucleus
dorsal raphé nucleus
sublemniscal prepacemaker nucleus
anterior tuberal nucleus
basal hypothalamic tract
optic tectum
posterior tuberal nucleus
periventricular nucleus of the posterior tuberculum
torus semicircularis, dorsal subdivision
torus semicircularis, ventral subdivision
ventral telencephalon, central nucleus
ventral telencephalon, dorsal nucleus
ventral telencephalon, intermediate nucleus
ventral telencephalon, dorsointermediate nucleus
ventral telencephalon, rostrointermediate nucleus
ventral telencephalon, lateral nucleus
ventromedial thalamic nucleus
ventral telencephalon, posterior nucleus
ventral telencephalon, supracommissural nucleus
ventral telencephalon, ventral nucleus
ventral telencephalon, ventral nucleus, dorsal subdivision
ventral telencephalon, ventral nucleus, ventral subdivision
nucleus of the zona limitans (thalami)
BASAL FOREBRAIN CONNECTIONS IN GYMNOTIFORM FISH
and sagittal sections (15-µm paraffin) of brains, either
stained with the Nissl stain, cresyl violet, or with KlüverBerrera to reveal fiber tracts and a cresyl violet counterstain to reveal cell bodies. Because all experimental material consisted of 50-µm sections cut with a Vibratome and,
therefore, had considerably different tissue shrinkage
from paraffin material, the anatomical organization of the
forebrain was further examined by using numerous cases
of material (50-µm Vibratome sections) from other experiments that did not involve label of basal forebrain nuclei.
These brains were counterstained with the Nissl stain,
neutral red (Sigma Chemical Co., St. Louis, MO). The
nomenclature used in the present study is that presented
in the whole brain atlas of the related gymnotiform
Apteronotus leptorhynchus (Maler et al., 1991). To confirm
that the corresponding cell groups in both taxa were being
assigned the same name, transverse sections of A. leptorhynchus were also studied.
Immunohistochemistry
To clarify further the anatomical organization of the
basal forebrain region, an antibody to the calcium binding
protein, calretinin, was used. Calretinin immunoreactivity
has been shown to be differentially distributed among
different populations of neurons (Baimbridge et al., 1992),
particularly in the forebrain of other vertebrates (e.g.,
Jacobowitz and Winsky, 1991) and was, therefore, used as
an additional tool for distinguishing cell groups of the
ventral telencephalon.
Recent biochemical studies in mormyrid and gymnarchid fishes identified an epitope recognized by this antibody, that is of the same molecular weight as mammalian
calretinin and not calbindin (Friedman and Kawasaki,
1997). Although it is likely that this protein is calretinin,
no functional role, in terms of calcium binding, or implication of homology with calretinin-like immunoreactive (CRl-ir) structures in other vertebrates will be considered in
its present usage. It is stressed that the usage of this
antibody is as a differential marker, like a Nissl stain or
many histochemical stains.
Three adult E. virescens were used for immunohistochemical studies. Animals were deeply anesthetized in
tricaine methane sulfonate (MS-222; Sigma) and perfused
transcardially with saline, followed by 30 ml of cold (4°C)
4% paraformaldehyde in 0.1 M phosphate buffer. Brains
were removed and post-fixed at 4°C. Free-floating Vibratome sections (50 µm) were cut into 0.02 M phosphate
buffered saline (PBS, pH 7.6) and rinsed three times, for 10
minutes each, in PBS. After rinsing, sections were placed
in a blocking solution of 5% normal goat serum (NGS;
Vector Laboratories, Burlingame, CA) in PBS with 0.3%
Triton X-100 (PBST) for 1 hour. After blocking, sections
were transferred to a 1% NGS solution in PBST containing
the primary antibody (rabbit anti-calretinin antibody;
SWant, Bellinzona, Switzerland) at 1:12,000 dilution. Sections were incubated for 2 days at 4°C in the primary
antibody solution.
After incubation, sections were rinsed three times, for 10
minutes each, in PBS, and then transferred to the secondary antibody solution. This consisted of 1% NGS in PBST
with a biotinylated goat anti-rabbit IgG (Vector) at 1:300
dilution. After 2 hours of incubation in secondary antibody,
sections were rinsed three times for 10 minutes in PBS,
then reacted by the avidin-biotin/peroxidase protocol de-
51
scribed below. Alternate sections were used for immunohistochemistry and normal anatomy.
By using a Vectastain avidin-biotin preoxidase complex
kit (ABC; Vector), an ABC solution was prepared (4 drops
A 1 4 drops B in 12 ml PBST). After 30 minutes of
prebinding of the ABC solution, sections were incubated in
the ABC solution in small, covered dishes at 4oC for 1 hour.
Sections were then washed three times for 10 minutes in
PBS and once for 10 minutes in Tris buffer (TB; 0.1 M, pH
7.2), then processed by the peroxidase/3,3(-diaminobenzidine (DAB) procedure. Sections were presoaked for 15
minutes in a solution of TB, and 0.04% DAB (Sigma). After
presoak, H2O2 was added to a final concentration of
0.0018%. The reaction was allowed to proceed for 5 to 15
minutes, depending on the intensity of the background
label. The reaction was stopped by washes in TB. Sections
were washed at least three times for 10 minutes in TB and
mounted on chrom-alum gelatin-coated slides.
Tract tracing
A total of 18 animals was used for tract tracing studies.
Injections were made into the ventral telencephalon (6
cases), the preoptic area (2 cases), the hypothalamic nuclei
(4 cases), and the nucleus electrosensorius (nE; 6 cases).
Over 40 additional cases of injections into other structures
(dorsal telencephalon, thalamus, torus, tectum, midbrain
tegmentum) were analyzed as controls for label attributable to spread. Technical considerations of tract-tracing
with Neurobiotin in E. virescens, particularly with regard
to transneuronal transport, have been described in detail
in a previous study (Wong, 1997a). Processing of tissue for
Neurobiotin tract tracing was from a protocol modified
from previous procedures (Kita and Armstrong, 1991;
Lapper and Bolam, 1991; Huang et al., 1992).
Anesthesia consisted of placing the fish in a 1:15,000
solution of MS-222 until somatic reactions stopped. This
procedure was followed by immobilization with an intramuscular injection (2 µl of 2 mg/ml in saline) of the
cholinergic blocker gallamine triethiodide (Sigma). The
fish was then placed in a fish holder with aerated water
perfusing the gills. The tail was placed in a plastic tube,
and a wire inserted in the tube so that the EOD could be
amplified and monitored. The surgical site was prepared
with a topical application of 2 mg/ml lidocaine (Veterinary
Companies of America, Tempe, AZ), and the skull was
opened to expose the rostral pole of the brain.
Injections were placed in brain nuclei, based on stereotaxic reference to the CP/PPn, which was initially localized
by iontophoresing the excitatory amino acid, L-glutamate,
and monitoring changes in the EOD (Kawasaki et al.,
1988; Keller et al., 1990; Heiligenberg et al., 1991; Heiligenberg et al., 1996). As EOD modulations could be elicited by
iontophoresis of L-glutamate into the nE, this nucleus was
identified by its direct stimulation (Keller and Heiligenberg, 1989). The single-barrel electrode was replaced with
a triple-barrel electrode consisting of two barrels filled
with glutamate and one with the tracer, Neurobiotin
(Vector). The Neurobiotin was made up at 2% in 0.2 µm
filtered 1 M KCl. Tip resistances for the Neurobiotin
electrodes were approximately 5 MOhms. Upon reidentification of the site, tracer was iontophoresed with
positive DC current (ca. 12 µA, 10 seconds on, 15 seconds
off) for 20 minutes up to 1 hour. After iontophoresis of
Neurobiotin, the electrode was left in place for 5 to 10
minutes, and then slowly withdrawn with backing current
52
C.J.H. WONG
to avoid leakage. The hole in the skull was patched with
Gelfoam (Upjohn Company, Kalamazoo, MI), and the site
was sealed with Vetbond (3M, St. Paul, MN). Survival
times were 8 to 30 hours at 27°C for retrograde and
anterograde transport of tracer.
Fish were killed in MS-222 and perfused transcardially
with 0.9% NaCl followed by 30 ml of fixative. The fixative
consisted of 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were then
removed and post-fixed overnight. Free-floating Vibratome sections were processed by using the ABC method
described above, with the following differences in the
protocol: (1) after sectioning, sections were prebleached in
0.5% H2O2 in PBS for 10 minutes to inhibit endogenous
peroxidases in the tissue; (2) sections were incubated in
ABC solution overnight at 4°C; (3) additional intensification with 0.064% nickel ammonium sulfate was used; (4)
every section was reacted, and subsequently counterstained, with neutral red.
Chartings of representative cases were made by using a
camera lucida. Brightfield photomicrographs were taken
by using an Olympus BH-2 photomicroscope, with TMAX100 black and white film (Kodak, Rochester, NY). Contrast
enhancement filters used for photomicrography were a
Wratten 44 filter to enhance the counterstain and neutral
density filters to adjust exposure. Prints were made on
Kodak multigrade resin-coated (RC) paper or Ilford multigrade RC paper (Ilford Photo, Paramus, NJ).
RESULTS
Cytoarchitectural organization
The midline nuclei of the basal forebrain of E. virescens
are a complex series of nuclei, bound laterally by the
forebrain bundle. The basal forebrain consists of several
distinctive subdivisions. The nomenclature used for the
telencephalon of ray-finned fishes is that of Nieuwenhuys
(1963), as adapted to a related gymnotiform fish, A.
leptorhynchus (Maler et al., 1991). Under this nomenclature, the telencephalon is parceled into a dorsal telencephalon (D) and a ventral telencephalon (V), with individual
nuclei given positional descriptors. Generally, the dorsal
telencephalon is recognized as comprising the pallium,
whereas the ventral telencephalon is the subpallium. In
the present study, I will address only the ventral nucleus of
the ventral telencephalon (Vv) and its immediate surround.
Precommissural regions. The Vv is a large, complex
nucleus situated at the base of the telencephalon. At
rostral levels (Fig. 1A1,B1), along the midline, the Vv can
be observed as a wing-shaped nucleus extending dorsolaterally from the ependymal zone. The Vv extends from the
dorsal, caudal aspect of the olfactory bulb to the level of the
anterior commissure. It is capped by the dorsal nucleus of
the ventral telencephalon (Vd), which can be observed as a
series of cell laminae, appearing as arches parallel to the
ventricular wall. The lateral extent of the Vv is bound by
the forebrain bundle (FB). Dorsolateral to the Vv, a
cup-shaped group of darkly staining cells, termed the
central nucleus of the ventral telencephalon (Vc) can be
recognized. At the lateral edge of the ventral telencephalon, a diffuse sheet of cells can be observed as the lateral
nucleus of the ventral telencephalon (Vl).
The Vv can be divided into a ventral subnucleus (Vv-v)
and a dorsal subnucleus (Vv-d). The dorsal subnucleus is
observed as a cigar-shaped stream of relatively small (5–8
µm), lightly staining, round cells, extending dorsolaterally
from the ventricular wall. The Vv-d can be distinguished
from the Vv-v, which generally appears as a more heterogeneous aggregate.
Postcommissural regions. Further caudally (Fig.
1C1), the Vv is flanked along its ventral aspect by the
supracommissural nucleus (Vs) that appears as a bed
nucleus of the anterior commissure (AC). Because the
supracommissural nucleus is intermingled with fibers of
the anterior commissure and is bound rostrally by the
bifurcation of a prominent blood vessel, the transition
between the posterior Vv and anterior Vs can be difficult to
discern. The Vs can be distinguished from the Vv in E.
virescens, as consisting of slightly larger (10 µm) and
diffusely scattered cells that stain relatively poorly for
Nissl substance. In the present description, the Vs is
considered to consist of both the region immediately dorsal
to the commissure and the loosely scattered elements,
situated around the horn of the commissure.
At commissural and postcommissural levels, the posterior nucleus of the ventral telencephalon (Vp) can be
observed dorsal to the Vd (Fig. 1C1,D1). The Vp can be
identified as a ventrolaterally extending sheet of darkly
staining cells. A loosely laminated, triangular region of
smaller cells is observed ventromedial to the Vp and has
been traditionally considered as part of the Vp (Maler et
al., 1991).
The Vv and Vs are replaced by the intermediate nucleus
of the ventral telencephalon (Vi), characterized as scattered, diffuse elements extending dorsolaterally from the
ventricular wall (Fig. 1D1).
Ventral to the anterior commissure, the anterior periventricular preoptic nucleus (PPa) can be observed as a
densely staining, tightly clustered, peri- and paraventricular cell group, bound by a lateral neuropil that is continuous with the medial forebrain bundle (Fig. 1C1). The
caudal aspect of the PPa is displaced laterally by the
laminar sheets of cells of the posterior periventricular
preoptic nucleus (PPp) which is capped by the Vi (Fig.
1D1). Caudal to the PPp, the anterior hypothalamic nucleus
(Ha) can be observed to eventually replace the dorsal
aspect of the PPp (not shown).
Calretinin immunohistochemistry
The basal forebrain nuclei are richly endowed with
calretinin-like immunoreactivity (CR-l-ir). In particular,
the Vv, Vl, and Vc display deeply staining CR-l-ir perikarya (Fig. 1A2,B2). In contrast, the Vd displays a diffuse,
terminal field of CR-l-ir with few immunoreactive cell
bodies. The subregions of Vv can be recognized further by
using CR-l-ir. The Vv-d appears as a dense cluster of
immunoreactive perikarya, in contrast to the Vv-v, which
has only a few scattered cells among a rich plexus of
CR-l-ir fibers and terminals.
At the level of the anterior commissure, CR-l-ir cells in
the Vv are bound ventrally by the relatively CR-l-ir
cell-poor supracommissural nucleus (Fig. 1C2). These are
eventually replaced by the Vi, which, in general, is lacking
in CR-l-ir, except for an occasional cell (Fig. 1D2). However, the Vp, can be recognized as having a dense meshwork of fibers, similar to the Vd. The PPa consists of a
sheet of heavily immunoreactive cells that appear rostrally near the ventricle and are displaced caudolaterally
by the PPp (Fig. 1C2, D2). The most rostral aspect of the
BASAL FOREBRAIN CONNECTIONS IN GYMNOTIFORM FISH
53
Fig. 1. Cytoarchitecture and calretinin-like immunoreactivity of
the basal forebrain of Eigenmannia virescens. A1,B1,C1,D1: Nisslstained transverse sections through the ventral telencephalon and
preoptic area (50-µm thick, 200-µm intervals). A2,B2,C2,D2: Calreti-
nin-like immunoreactivity in the section immediately caudal to the
section presented in the left hand column. For abbreviations, see list.
Scale bar 5 200 µm in D2 (applies to A1–D2).
PPp appears to have few CR-l-ir cells, whereas the transitional zone with the Ha, appears to contain lightly immunoreactive cell bodies (not shown).
the ventromedial aspect of the bulb constituting the terminal nerve ganglion (not shown).
Telencephalon. In the dorsal telencephalon, retrogradely labeled cells were consistently observed in the
posterior nucleus (Dp). These bipolar and multipolar cells
were generally confined to the medial aspect of the Dp (Fig.
2F). In the ventral telencephalon, retrogradely labeled
somata were observed ipsilateral to the injection site
within the Vs (Figs. 2D, 3B). More caudally, a band of cells
could be observed extending throughout the most caudal
aspect of the dorsal intermediate nucleus (Vid; Figs. 2F,
4C). Labeled somata were not observed in the entopeduncular nuclei or other subnuclei of the ventral telencephalon with injections confined to the Vv.
A bilateral projection was also observed from a sheet of
cells of the nucleus taenia (nT), situated along the telencephalo-diencephalic junction (Figs. 2F, 4C). Rostrally, the
nT appears immediately medial to the caudal entopeduncular nucleus. At more caudal levels, the labeled cells
within nT cap the caudal entopeduncular nucleus and
eventually appear subadjacent to the Dp.
Afferents to the Vv
Injections were placed in the rostral Vv. Of the six
injections placed into the Vv, only three injection sites
were discretely confined to the Vv without significant
encroachment on adjacent areas. Figure 2 documents a
global injection centered on and mostly confined to Vv.
Although, in the following description, I present the extent
of labeled cells and fibers from this global injection (Fig.
3A), cell groups considered afferents or efferents (Table 1)
were assessed by smaller, confined injections and control
injections. Most connections to and from the Vv were primarily
ipsilateral with a weaker contralateral component.
Olfactory bulb. The most rostral extent of labeled
cells were mitral cells within the medial aspect of the
olfactory bulb (Figs. 2A, 4A). In contrast, labeled cells were
not observed in the lateral portion of the bulb after
injections that were strictly confined to Vv. Additionally,
relatively large cell bodies were observed bilaterally along
Fig. 2. A–I: Camera lucida drawings of representative sections of
label after a global injection of Neurobiotin centered on the ventral
nucleus of the ventral telencephalon (Vv). Large dots indicate
retrogradely labeled cell bodies; small dots indicate terminal fields.
Broken lines indicate fibers. Asterisks in G, H indicate the zona
limitans. Inset: side view of the brain of E. virescens. Numbers
indicate the corresponding level for this figure and Figure 1. Fb,
forebrain; Cb, cerebellum; ELL, electrosensory lateral line lobe. For
abbreviations, see list. Scale bar 5 200 µm in I (applies to A–I), 1 mm
in inset.
BASAL FOREBRAIN CONNECTIONS IN GYMNOTIFORM FISH
55
Fig. 3. A: Photomicrograph of a Neurobiotin injection site into the Vv charted in Figure 2C. B: Labeled
cells and fibers observed at the level of the anterior commissure. Note the heavy fiber label both within the
anterior periventricular preoptic area (PPa) and the supracommissural nucleus (Vs). For abbreviations,
see list. Scale bars 5 100 µm.
Less consistently observed were labeled cells in the
rostral-most region of the central nucleus of the dorsal
telencephalon (Dc; Fig. 2B,C). Due to the proximity of the
labeled cells in Dc to the rostral injection sites and that
this cell group was not observed to be labeled with very
discrete injections, it is likely that the labeled somata in Dc
constitute spread of tracer from the injection site.
Diencephalon and mesencephalon. Caudally, labeled cells were observed throughout the medial aspect of
the preoptic area (Figs. 2D,E, 3B). Retrogradely labeled
somata were observed primarily throughout the anterior
periventricular preoptic nucleus (PPa) and the posterior
periventricular preoptic nucleus (PPp). In contrast, more
caudal aspects of the preoptic region, including the anterior hypothalamic nucleus (Ha) and anterior periventricular nucleus (nAPv), contained few retrogradely labeled
somata (Fig. 2F). The lateral (Hl), ventral (Hv), and caudal
(Hc) hypothalamic nuclei, and the anterior portion of
lateral tuberal nucleus (nLTa) contained numerous labeled somata (Figs. 2G,H,I, 4D). Occasionally, a few cells
were observed within the lateral nucleus of the lateral
recess (nRLl). Other major afferent cell groups observed
include the central posterior thalamic nucleus/prepacemaker nucleus (CP/PPn), the posterior tubercular nucleus
(TP), and the locus coeruleus (LCe; not shown). Occasionally, cells were observed in the dorsal posterior thalamic
nucleus (DPn), the ventral thalamus, the periventricular
nucleus of the posterior tuberculum (TPP) and an isthmic
TABLE 1. Connections of the Ventral Nucleus of the Ventral
Telencephalon1
Eigenmannia
virescens
(this study)
Afferents to the Vv
Olfactory bulb
Telencephalon
Dorsal Telencephalon
Ventral Telencephalon
Diencephalon
Hypothalamus
Thalamus
Posterior tuberculum
Mesencephalon
Olfactory bulb
Telencephalon
Diencephalon
Hypothalamus
Thalamus
Pretectum
1For
Carassius
auratus
(Sloan, 1989)
Afferents to the Vv/Vs
Terminal nerve, medial
olfactory bulb
Terminal nerve, olfactory
bulb
Dpm
Vs, Vid, nT
Dc, Dl
Vc, Vd, Vp, Vi-nT
PPa, PPp
Ha, nAPv (minor)
Hl, Hv, Hc
nLTa, nRLl (minor)
CP/PPn
DPn (minor)
VMTh (minor)
TPP, TP
LCe
PLm
Efferents from the Vv2
Medial olfactory bulb
Vs (Vi, minor)
PPa
PPa, PPp (Ha, Hv, Hl, nRLl,
Hc)
DPn
DP/PPn
nE< (rest of nE, minor)
Hd, Hc
CP
DPn
TPP, TP
LCe
Raphe
Efferents from the Vv/Vs
Vc
Dc, Dl
PPa
Habenula
abbreviations, see list.
2Efferents indicate observed terminal fields. Due to label of axon collaterals and
difficulty in mapping the precise topography with the hypothalamic nuclei, efferents
that were not clearly identified are indicated with brackets.
56
C.J.H. WONG
Fig. 4. Photomicrographs of afferents to the Vv. A: Retrogradely
labeled mitral cells in the medial olfactory bulb (differential interference contrast optics). B: Retrogradely labeled somata in the dorsal
intermediate nucleus of the ventral telencephalon (Vid). C: Labeled
somata in nucleus taenia (nT). Note the heavy fiber label in the medial
forebrain bundle (MFB; arrowheads) in contrast to the lateral forebrain bundle. D: Labeled cells (arrowheads) and fibers in the ventral
hypothalamic nucleus (Hv) and lateral hypothalamic nucleus (Hl) in
contrast to the anterior tuberal nucleus (TA). For abbreviations, see
list. Scale bars 5 50 µm in A–C, 200 µm in D.
nucleus, the medial perilemniscal nucleus (PLm; Maler et
al., 1991).
could be traced ventrally and caudally to innervate the
preoptic area and a few fibers were observed to extend
dorsolaterally toward the rostral aspect of the ventrolateral nucleus of the dorsal telencephalon (Dlv). However,
this projection to the Dlv was very weak and not observed
with confined injections into the Vv. A few fibers were also
observed within the rostral entopeduncular nucleus (Fig.
3B).
A dense plexus of terminals and fibers was observed in
both the medial cellular region of the rostral preoptic area
(PPa, PPp) and the lateral neuropil, including the medial
forebrain bundle, which can be observed medial to the
entopeduncular nucleus (Figs. 2D,E,3B). Further caudally,
fibers could be traced within the medial forebrain bundle
(MFB) to the transverse level of the anterior hypothalamus (Fig. 4C). At this level, a dorsal band of fibers (Fig. 2F)
was observed to continue caudally to innervate the thalamus, in particular the dorsal neuropil between the CP/PPn
and the DPn (Fig. 2H). A second band of labeled fibers
appeared to course ventrally and interweave with the
commissural fibers of the postoptic commissure (Fig. 2F).
Two separate components of this band could be traced. A
Efferents from the Vv
After injections into Vv, anterogradely labeled fibers
were observed, coextensive with many cell groups that
contained retrogradely labeled somata. However, as Neurobiotin is transported in both retrograde and anterograde
directions, it is unclear as to what degree labeled fibers
constitute efferents from the Vv or anterograde label of
collaterals of afferents to the Vv. Therefore, the description
of labeled fibers will be presented followed by descriptions
of reciprocally placed injections. The extent by which
labeled fibers can be attributed to labeled efferent cells of
the Vv, and not labeled collaterals of afferents to the Vv,
are derived from these injections.
Rostrally, labeled fibers were observed to exit the injection site toward the olfactory bulb. Caudally, labeled fibers
were observed to exit from the injection site to the level of
the commissure of Goldstein, where many of the fibers
appeared to decussate, innervating homotopic structures
of the contralateral hemisphere. However, some fibers
BASAL FOREBRAIN CONNECTIONS IN GYMNOTIFORM FISH
57
Fig. 5. Anterogradely labeled fibers and terminals in the pretectal
nucleus electrosensorius, down subdivision (nE<). Scale bar 5 50 µm.
lateral component appeared to course dorsal to the preglomerular complex and innervate the pretectum (Figs. 2G,
5). A medial component could be traced along the lateral
edge of the anterior tuberal nucleus to a prominent
terminal field in the lateral and caudal hypothalamic
nuclei (Figs. 2G,H,I, 4D). Some fibers continue caudally
through the basal hypothalamic tract (tBH) toward the
pituitary (not shown).
A band of fibers could be observed ventral to the thalamus, near the zona limitans (Fig. 2G,H). This band of very
fine labeled fibers could be traced along the dorsal aspect of
the lateral recess to a terminal field in the lateral nucleus
of the lateral recess (nRLl).
Injections into the PPa/PPp
Because heavily labeled cells and terminals were observed in the ipsilateral PPa/PPp, after discrete injections
into Vv, two injections were made into the rostral preoptic
area to further examine the organization of efferents from
Vv. These injections were not exclusively confined to the
PPa or PPp and hence will be described briefly. Injections
centered into PPa/PPp retrogradely labeled many cell
groups, similar to what was observed after injections into
Vv. In considering these regions in which labeled cells were
observed from both injections into the Vv and the PPa/
PPp, it is unclear as to what degree these cell groups
project to both the Vv and the PPa/PPp, or the Vv only. It is
possible that subsequent spread of tracer into the forebrain bundle from injections into the PPa/PPp might
explain overlapping patterns in labeled afferents. Of these
caudal regions that were observed to be retrogradely
labeled from both injections into Vv and the PPa/PPp, only
connections between the PPa/PPp with the CP/PPn were
fully corroborated (Wong, 1997a).
After injections of Neurobiotin centered in the PPa/PPp
(Fig. 6), retrogradely labeled somata were observed both
within the Vv-v and the Vv-d. Anterogradely labeled fibers
were also observed in both Vv-v and Vv-d; however, labeled
terminals appeared to be more prominent in the Vv-v (Fig.
7A).
Two cell groups were identified that had retrogradely
labeled cell bodies uniquely from injections into the PPa/
PPp and not injections confined to the Vv. One afferent cell
group to the PPa/PPp was the dorsal hypothalamic nucleus
(Hd; Fig. 7B). A second afferent cell group that was
Fig. 6.
Injection site into the preoptic area centered at the
interface of the anterior and posterior periventricular preoptic nuclei
(PPa/PPp). White arrow indicates electrode tracking. For abbreviations, see list. Scale bar 5 100 µm.
observed was located in the midbrain tegmentum in the
vicinity of the SPPn (Fig. 7D). These multipolar tegmental
neurons were similar in morphology to those that were
typically labeled after injections into the pacemaker; however, they were situated ventrolateral to the SPPn (indicated by asterisks in Fig. 7D). This finding suggests that
these cells comprise a distinct subregion of the midbrain
tegmentum and are not SPPn neurons proper. Similar to
injections into the Vv, numerous anterogradely labeled
terminals were observed in the CP/PPn coextensive with
labeled cell bodies (Fig. 7C).
Injections into the hypothalamic nuclei
Injections were made in the hypothalamic nuclei to test
hypotheses of connectivity with the preoptic area and the
Vv. However, because none of the injections placed were
specifically confined to a single hypothalamic nucleus and
that fibers originating from hypophysiotrophic cell groups
in the Vv and PPa/PPp course through the basal hypothalamic tract (Johnston and Maler, 1992), the specific organization of forebrain afferents to these various hypothalamic
nuclei remains unknown. Briefly, after injections centered
into the lateral hypothalamic nucleus encroaching on the
anterior tuberal nucleus and nucleus of the lateral recess,
both labeled cell bodies and fibers were observed in both
subdivisions of the Vv and throughout the preoptic area.
58
C.J.H. WONG
Fig. 8.
A: An injection of Neurobiotin, centered on, but not
confined to, the nE<. B: Observed retrogradely labeled cells in the Vv-v.
Inset: High-powered photomicrograph of cells in Vv-v. Scale bars 5
100 µm in A,B, 25 µm in the inset.
Injections into the nucleus electrosensorius
As described above, a minor efferent projection was
observed from the Vv to the pretectal nucleus electrosensorius. Although an occasional fiber was observed in most
subdivisions (not shown), the most prominent pretectal
target of the Vv was the ‘down’subdivision of the nE (nE<),
and this projection was minor in comparison to fibers
distributed through the thalamus, preoptic area, and
hypothalamic nuclei. Because the nE< could be located by
glutamate stimulation (Keller and Heiligenberg, 1989),
precise injections were placed in this nucleus, and the
presence of this connection was tested (Fig. 8A). Retrograde transport of Neurobiotin confirmed this minor projection and revealed that the cell bodies of origin were
situated in the Vv-v (Fig. 8B).
Control injections
Fig. 7. Labeled cells and fibers after an injection in the rostral
preoptic area. A: Labeled cells and fibers in Vv. Note the heavier
innervation of the ventral subdivision of Vv (Vv-v). B: Retrogradely
labeled somata in the dorsal hypothalamic nucleus. C: Retrogradely
labeled somata and anterogradely labeled fibers in the dorsal aspect of
the medial CP/PPn. Note terminal swellings within the CP/PPn
(arrows). D: Retrogradely labeled somata (arrows) ventrolateral to the
sublemniscal prepacemaker nucleus. Asterisks indicate boundaries of
the SPPn. For abbreviations, see list. Scale bars 5 100 µm in A, 25 µm
in B, 50 µm in C, 100 µm in D.
Further work will be necessary to clarify the precise
topographic organization of connections that the Vv and
PPa/PPp share with individual hypothalamic nuclei.
Due to the depth of the Vv and PPa/PPp, one major
concern is that injections into these structures might have
encroached onto overlying nuclei. Only three injections
into the Vv were small enough so as to be specifically
confined to the Vv, without any significant encroachment
on the rest of the telencephalon. Therefore, subtractive
analysis was used to further consider hypotheses of connections for larger injections. Control injections were placed
into the telencephalon such that Vv or the PPa were
specifically excluded from the injection site. The observed
pattern of labeling is briefly described below.
One control injection consisted of an injection centered
on the overlying pallium, in the second subdivision of the
dorsomedial telencephalon (Dm2; Fig. 9A). In this case,
there were no labeled cells observed in the CP/PPn, nor
fibers observed in the nE or medial preoptic area. Anterogradely labeled fibers were observed within the most
lateral aspect of the lateral hypothalamic nucleus. However, the ventral hypothalamic nucleus as well as more
medial portions of the lateral hypothalamic nucleus in
which labeled fibers and cells were more typically observed
after discrete injections into Vv or the PPa (e.g., Fig. 4D),
were not observed to be labeled with an injection centered
on the Dm2.
A second control injection consisted of a large injection of
Neurobiotin, centered on, although not confined to, the Vd
with only minor encroachment on the Vv (Fig. 9B). In this
BASAL FOREBRAIN CONNECTIONS IN GYMNOTIFORM FISH
59
Fig. 9. Camera lucida drawings of the center of injection sites for control injections. See text for
details. A: Injection of Neurobiotin centered on the second subdivision of the dorsomedial telencephalon
(Dm2). B: Injection of Neurobiotin centered on, although not confined to, the dorsal nucleus of the ventral
telencephalon (Vd). For abbreviations, see list. Scale bar 5 200 µm.
case, there were only a few labeled somata observed in the
preoptic area, the CP/PPn, and the nT, which contrasted
with injections that were centered more ventrally. There
were numerous labeled cells in the intermediate nucleus of
the ventral telencephalon; however, these were accompanied by dense labeling of fibers, in contrast to injections
centered in the Vv in which there was very sparse anterograde labeling of fibers in the Vi. Additionally there were
no retrogradely labeled somata observed in the olfactory
bulb, the Vs, or the hypothalamic nuclei.
Retrogradely labeled cells were observed in the central
nucleus of the dorsal telencephalon, after injections of
tracer into the torus semicircularis and optic tectum.
Therefore, it is unclear as to whether observed labeled
cells in Dc actually project to Vv or whether interruption of
fibers of passage may account for labeled cells in Dc.
DISCUSSION
The results of the present study are summarized in
Figures 10, 11, and in Table 1. The ventral nucleus of the
ventral telencephalon of E. virescens (Vv) is heavily and
reciprocally connected with the rostral nuclei of the preoptic area (PPa, PPp), the hypothalamic nuclei (nLTa, Hl, Hv,
Hc, nLTa, nRLl), and as reported previously (Wong, 1997a),
the thalamus (CP/PPn). Other inputs originate from the
olfactory bulb and terminal nerve, caudal telencephalon
(Dpm, Vid, nT), posterior tuberculum (TPP, TP), the locus
coeruleus (LCe), and the medial perilemniscal nucleus
(PLm). Moreover, subdivisions of the Vv can be recognized
based upon cytoarchitectonic, chemoarchitectonic, and connectional criteria.
Subdivisions of the ventral nucleus of the
ventral telencephalon
The present study recognizes all the major subdivisions
of the ventral telencephalon and rostral preoptic area that
have been identified in other teleosts (see Northcutt and
Braford, 1980; Northcutt and Davis, 1983, for a review). In
E. virescens, the Vv consists of at least two recognizable
subdivisions. Only one other study, that of Bass (1981a) on
the channel catfish, Ictalurus punctatus specifically addresses subdivisions of Vv and the present interpretations
differ somewhat from that of Bass (1981a). The two
subdivisions of Vv identified by Bass (1981a) cannot be
Fig. 10.
Organization of connections that are topographically
organized with respect to the dorsal (Vv-d) and ventral subdivisions of
the Vv (Vv-v). A: Afferent projections to the Vv. Relative strength of
projections is indicated by thickness of the lines. B: Efferent projections of the Vv. Same presentation as in A. Other projections for which
the topographic organization is unknown are listed in Table 1. See text
for details. For abbreviations, see list.
recognized in E. virescens, and therefore the Vv-v in E.
virescens likely corresponds to both subdivisions of Vv
recognized by Bass (1981a). Based on the position of
olfactory terminal fields in the related gymnotiform A.
leptorhynchus (see Sas et al., 1993, for details), it is likely
that the dorsal subnucleus of the Vv (Vv-d) in the present
study corresponds to the ventral subnucleus of Vd, recognized in the channel catfish (Vd-v; Bass, 1981a). It would
also appear to correspond to the Vs of Levine and Dethier
(1985), in the common goldfish, Carassius auratus. Although the secondary olfactory projections of E. virescens
were not examined in the present study, based on cytoarchitecture, similar subdivisions can be recognized to be
present in A. leptorhynchus.
In addition to cytoarchitectonic criteria, chemoarchitecture distinguishes subdivisions of the Vv. The Vv-d can be
60
C.J.H. WONG
recognized as being distinctive from both the Vv-v and the
Vd in having tightly clustered CR-l-ir perikarya. This
contrasts with the Vv-v, which has a high density of
periventricular immunoreactive somata and only scattered CR-l-ir somata laterally. Moreover, it also contrasts
with the Vd, which contains primarily meshwork of fine,
immunoreactive fibers and terminals. In A. leptorhynchus,
the Vv-d is extremely rich in substance-P immunoreactive
fibers and terminals in contrast to the Vv-v and the Vd
(Weld and Maler, 1992). Moreover, the Vv-d appears to be
more heavily innervated by enkephalinergic fibers (Richards and Maler, 1996), further corroborating the heterogeneous nature of the Vv.
Connectional evidence distinguishes the two subdivisions of the Vv. The Vv-d and to a lesser degree, the ventral
aspect of the Vd, share prominent reciprocal connections
with the thalamic CP/PPn (see Fig. 3a of Wong, 1997a) and
appear to overlap with the medial olfactory terminal field
(Sas et al., 1993). Moreover, the Vv-d provides a source of
afferents to the olfactory bulb (Sas et al., 1993). In contrast, the Vv-v receives heavy input from the PPa/PPp,
whereas the Vv-d receives a somewhat weaker projection.
The Vv-v appears to be only weakly connected with the
thalamus (Wong, 1997a).
Consideration of the transitional zone between the Vv
and the Vs is more problematic. The original cytoarchitectonic description of Vs was merely as the supracommissural extension of the Vd (Nieuwenhuys, 1963). Therefore,
the Vs would be situated dorsal and caudal to the Vv
(Northcutt and Braford, 1980; Northcutt and Davis, 1983).
However, reexamination of the application of the term Vs
to teleosts (Nieuwenhuys, 1963) reveals that this designation grouped together several nuclei that had been recognized as distinctive by previous authors.
The present study considers the Vs as a bed nucleus of
lightly staining cells situated ventral to the Vv (see
Results). The location is in agreement with the atlas of the
related gymnotiform A. leptorhynchus (Maler et al., 1991)
as well as the atlas of the goldfish (Peter and Gill, 1975). It
would appear to correspond to the bed nucleus of the
commissura hippocampi of Schnitzlein (1962).
Technical considerations to tract-tracing
experiments
The Vv receives input from the olfactory bulb (Bass,
1981b; Levine and Dethier, 1985; Sas et al., 1993; Matz,
1995) and has cells that project to the pituitary (Johnston
and Maler, 1992). Due to the proximity of the injection
sites to the forebrain bundle, one major concern is that
damaged fibers of passage might have taken up tracer.
Therefore, I discuss the following considerations of alternative hypotheses regarding observed patterns of labeled cell
groups.
Some cell groups, that were observed to be retrogradely
labeled after injections into the Vv, also project to the bulb.
Therefore, labeled cells might have resulted from interruption of bulbopetal fibers (e.g., Vs, Dpm, nT). However,
many cell groups that are afferent to the bulb were not
observed to be retrogradely labeled, after discrete injections of tracer into Vv. These include the Dc shell, the
rostral aspect of the Dp, and the dorsomedial telencephalon (Sas et al., 1993). These data suggest that the contribution of labeled cells, attributable to interruption of bulbopetal fibers, was probably minor.
Two major efferent targets of the Vv were the Vs and the
anterior preoptic area (PPa, PPp). The centromedial subdivision of the medial olfactory tract, that carries bulbofugal
fibers to the ventral telencephalon and possibly the preoptic area (Bass, 1981b; Levine and Dethier, 1985; although
see Sas et al., 1993; Matz, 1995), courses near the Vv. Thus
the bulbofugal fibers to these regions may have been
interrupted by the present injections. Evidence against
such an interpretation is as follows: (1) both the habenular
commissure and the anterior commissure carry commissural fibers of bulbofugal cells, however, generally lacked
any labeled fibers from injections confined to the Vv; (2)
many of the cell groups that receive bulbofugal input from
the medial olfactory tract, contained only very sparse label
of fibers or terminals (e.g., Vir, Vc); and (3) at supracommissural levels, the medial olfactory terminal field was observed to be relatively lightly labeled, possibly from labeling of collaterals, in contrast to the heavy terminal labeling
in the Vs and the PPa (e.g., Figs. 2D, 3B). The presence of
heavy anterogradely labeled fibers in the Vs, PPa, and
PPp, in contrast to what was observed in other bulbofugal
cell groups, after injections into Vv, suggests that the Vv
does project to the Vs, PPa, and PPp.
A last confound is that labeled cells and fibers observed
within the Vv, after injections into the PPa/PPp, may be
due to interruption of afferents from and efferents to Vv.
There are two pieces of evidence against such an interpretation. First, after injections into the PPa/PPp, most
anterogradely labeled fibers were observed within the
Vv-v, in contrast to the Vv-d. The Vv-d receives a heavy
thalamofugal projection (Wong, 1997a), and it is clear that
labeled fibers in the Vv-d were few in comparison, after
relatively confined injections into the PPa (Fig. 7A). A
second argument against such an interpretation is the
high density of cellular, terminal, and fiber labeling in the
PPa/PPp after injections into the Vv. This finding suggests
that the Vv and the PPa are heavily interconnected.
Because there was considerable overlap in cell groups
that were retrogradely labeled from injections either into
PPa or Vv (Dpm, nT, hypothalamic nuclei), it remains
unclear as to whether these nuclei project to Vv exclusively
or also project to the PPa/PPp in addition to the Vv.
Injections of anterograde tracer into these regions will be
necessary to delineate the full extent of overlap in afferent
cell groups to the basal forebrain and preoptic area.
Anterograde confirmation of thalamic afferents to both the
PPa/PPp and basal forebrain have been demonstrated
previously (Wong, 1997a). It is also possible that individual cells may project to both the Vv and PPa/PPp,
further coordinating these two areas. This hypothesis will
need to be tested by double-label retrograde studies.
Other concerns of spread were further tested by control
injections centered into overlying structures. These injections revealed very different patterns of labeled cells and
fibers in comparison to what was observed after injections
centered in the Vv (see Results). Specifically, labeled cells
were not observed in the preoptic area, ventral, lateral, or
caudal hypothalamic nuclei, nE, or CP/PPn. Labeled cells
were observed in the Dc after injections into the torus and
tectum, as has been reported in other teleost fishes (Ito
and Kishida, 1977; Murakami et al., 1983; Echteler, 1984).
Therefore, it is unclear whether Dc actually projects to the
Vv or whether interruption of fibers of passage may
account for the observed labeled cells.
BASAL FOREBRAIN CONNECTIONS IN GYMNOTIFORM FISH
Afferents to the ventral telencephalon
The connections identified in the present study are in
good agreement with those identified previously in the
goldfish (Sloan, 1989). Some connections have also been
identified in the land-locked salmon, although identification of correspondence between specific cell groups is
limited by differences in cytoarchitectonic delineation (Oncorhynchus nerka; Shiga et al., 1985a, 1985b). These
results are summarized in Table 1. It is possible that the
differences in connectivity may be due to species-specific
differences or methodological differences such as the sensitivity of the tracer used, and the size, placement, and
interpretation of injection sites.
In E. virescens, after injections of tracer confined to Vv,
retrogradely labeled cells were observed within the medial
aspect of the olfactory bulb. Only after injections that were
clearly not confined to the Vv were retrogradely labeled
cells observed in the lateral portion of the bulb. This
finding reinforces the notion that the olfactory bulb is a
heterogeneous structure consisting of at least a medial and
lateral subdivision based upon connectional (Bass, 1981b;
Levine and Dethier, 1985; Sas et al., 1993; Matz, 1995) and
immunohistochemical (Weld and Maler, 1992; Sas et al.,
1993) criteria. In the present study, retrogradely labeled
cells were observed bilaterally in the olfactory bulb, confirming the bilateral nature of bulbar efferents to the
ventral telencephalon (Bass, 1981b; Sas et al., 1993). In
contrast, in the common goldfish (Sloan, 1989), retrogradely labeled somata were observed in only the ipsilateral bulb after injections into the ventral telencephalon.
However, it is possible that the differences may be due to
the increased sensitivity of tracer detection for Neurobiotin versus the horseradish-peroxidase (HRP) tracing technique used previously (Sloan, 1989). Indeed, previous
investigators identified a bilateral bulbofugal projection to
Vv in the goldfish after injections into the olfactory bulb
(Levine and Dethier, 1985).
Several large round somata were retrogradely labeled
along the ventromedial aspect of the bulb. Although the
axons from these cells were not traced to the retina, it is
likely that these larger cells are ganglion cells of the
terminal nerve (Von Bartheld and Meyer, 1986).
Telencephalic afferents to the Vv in E. virescens are very
restricted (Vid, Vs, Dpm, and nT), although additional
afferents have been reported in the goldfish (Sloan, 1989;
Vd, Vc, Vp, Dl, Dc). However, as connections reported
previously include afferents to Vs (Sloan, 1989), it is
possible that the differences may be due to inclusion of the
Vs in injection sites in studies on the goldfish (Sloan,
1989). The present study does suggest that the Vv and Vs
are intimately related as has been described in other fishes
(Shiga et al., 1985a, 1985b; Sloan, 1989). Additionally, in
the present study, as labeled cells were not observed in Dc
after discrete injections into the Vv, labeled somata in the
rostral aspect of Dc are considered to be due to interruption of their descending fibers.
The Dp and the nT are also reciprocally connected with
the olfactory bulb. Although the specific functions of these
regions are unknown, the Dp, which is considered a major
olfacto-recipient area, is hypertrophied in gymnotiform
fish, which is surprising given the small size of the
olfactory bulb (Maler et al., 1991; Striedter, 1992; Sas et
al., 1993). Noting the hypertrophy of other structures
related to electrosensory/motor processing in gymnotiform
61
fish, it has been proposed that the Dp may play a role in
electroreception (Maler et al., 1991; Sas et al., 1993).
However, the existence of connections between the Dp and
electrosensory/motor areas remains to be determined.
The present study is in good agreement with previous
studies with regard to diencephalic afferent cell groups to
the Vv, although there may be some variation due to
differences in cytoarchitectonic descriptions and injection
placement. A major afferent projection from the preoptic
area to the Vv has been described in the goldfish (Sloan,
1989), as have inputs from the hypothalamic nuclei, the
dorsal thalamus, and the nucleus of the posterior tuberculum. Similarly, many of these projections (preoptic area,
dorsal thalamus, posterior tubercular nucleus) appear
similar to projections to the Vs in the land-locked salmon,
although it is likely that the Vv was also included in the
injection site (Shiga et al., 1985a).
In the channel catfish, Striedter (1991) observed labeled
cells and fibers in the Vv after application of tracer to the
TA. However, neither labeled cells nor fibers were observed
in the TA after injections into the ventral telencephalon in
any fish (Shiga et al., 1985a; Sloan, 1989; this study). It is
likely that this discrepancy is due to differences in interpretation of tracer spread. In both E. virescens (this study)
and the goldfish (Sloan, 1989), a number of hypothalamic
nuclei situated in close proximity to the TA are reciprocally
connected with the Vv. In contrast to what has been
reported in the goldfish (Sloan, 1989), projections with the
habenula were not observed in E. virescens. However, a
recent retrograde study in the goldfish showed that the Vd
projects to the habenula (Villani et al., 1996).
Functional considerations
Besides heavy interconnectivity with each other and
hypothalamic areas, the Vv and PPa/PPp of E. virescens
are interconnected with the electrocommunicatory system.
The Vv and preoptic area share reciprocal connections
with the rostral, medial aspect of the CP/PPn (Wong,
1997a). Moreover, many hypothalamic nuclei, that had
both labeled cells and fibers after injections of Neurobiotin
into the Vv (PPa, PPp, Hl, Hc), are also reciprocally
connected with the CP/PPn (pathway 1 in Fig. 11). These
results complement and extend previous immunohistochemical studies on a related gymnotiform, A. leptorhynchus, that identified extensive peptidergic and monoaminergic fibers in both the Vv and the CP/PPn whose cell
bodies of origin were hypothesized to be hypothalamic (see
Johnston and Maler, 1992, for a review). Moreover, many
of the cell groups identified to be connected with both the
Vv and the CP/PPn also have cells that project to the
pituitary (Johnston and Maler, 1992). Although further
studies will be necessary to determine specifically the
extent of overlap in individual cells of the hypothalamic
nuclei that are connected with the CP/PPn, the basal
forebrain, and the pituitary, the current study suggests
that the Vv, rostral preoptic area, lateral and caudal
hypothalamic nuclei, and CP/PPn constitute a network of
intimately associated cell groups.
A minor projection has also been identified from the Vv
to the pretectal nE. Previous studies reported observing
terminals in the nE, after massive injections of tracer into
the telencephalon (Keller et al., 1990). The present study
confirms this observation and identifies the cell bodies of
origin to be specifically the Vv-v.
62
C.J.H. WONG
Fig. 11. Relationship of cell groups, identified in the present study to be connected with the Vv (heavy
arrows), cell groups connected with the CP/PPn (1) and cell groups that project to the pituitary (2). See
text for details. For abbreviations, see list.
Although one or two fibers were observed in other
subnuclei of the nE, the most prominent subnucleus that
receives input from the Vv was the nE<. Stimulation of the
nE< in E. virescens elicits gradual decreases in EOD
frequency (Keller and Heiligenberg, 1989), mediated by an
inhibitory projection to the mesencephalic electromotor
nucleus, the SPPn (Metzner, 1993). Besides a role in the
jamming avoidance response (JAR) (Heiligenberg, 1991),
gradual decreases in frequency of the EOD appear to be
involved in social interactions in E. virescens (Hagedorn
and Heiligenberg, 1985). In other gymnotiform fishes, the
SPPn also mediates a variety of other modulations in the
EOD (Keller et al., 1991; Kawasaki and Heiligenberg,
1989; Spiro, 1994; Heiligenberg et al., 1996). Further work
will be necessary to determine the functional role of this
descending projection from Vv to the nE<.
The behavioral functions of the Vv and PPa have been
examined in detail in a variety of teleost fishes (reviewed
in Demski, 1983). In the goldfish, lesions of the posterior
Vv and Vs severely impair male spawning behavior (Kyle
and Peter, 1982; Kyle et al., 1982). Moreover, stimulation
studies in many fishes have shown that the rostral preoptic area may be involved in the production of courtship
displays (Demski and Knigge, 1971; Satou et al., 1984;
Fine and Perini, 1994).
Behavioral studies in E. virescens revealed that the
EOD, which is normally at a fixed frequency, is modulated
during courtship and agonistic encounters (Hopkins, 1974;
Hagedorn and Heiligenberg, 1985). One prominent modulation produced, primarily by male fish, are brief interruptions (ca. 10–100 ms) in the EOD, which may serve as
courtship signals to female fish. In E. virescens, electrical
stimulation of the preoptic area elicits interruptions in the
EOD (Wong, 1997b), suggesting that these basal forebrain
pathways may also be involved in the production of
electrocommunicatory signals.
Connections with the medial olfactory bulb lend further
credence to the notion that the basal forebrain may be
involved in reproductive/ communicatory behavior. The
medial olfactory tract, in the goldfish, has been implicated
in the relaying of sex pheromonal information to the
ventral telencephalon (see Dulka, 1993, for a review).
Therefore, it will be of interest to determine whether
gymnotiforms use pheromonal signals and if so, whether
the Vv-d and Vs might integrate such signals with ascending thalamic electrosensory information (Wong, 1997a).
Comparative aspects
The present connectional results do not clarify specific
homologies between the Vv and individual septal or basal
forebrain nuclei of other vertebrates, and it is possible that
such a one-to-one correspondence may not exist (Reiner
and Northcutt, 1992; Northcutt, 1995). However, the Vv of
E. virescens does share many connectional and functional
similarities to the septal region of other vertebrates.
One major cell group connected with the septal nuclei in
other vertebrates is the amygdala (amphibians: Neary and
Northcutt, 1990; rats: Krettek and Price, 1978; Swanson
and Cowan, 1979). Labeled cells and fibers were observed
within Vs, which may correspond to the amygdala, based
upon topological and chemoarchitectonic criteria (Reiner
and Northcutt, 1992; Northcutt, 1995; although, see Sas et
al., 1993; Weld et al., 1994). Another major afferent region
to the septum is the medial pallium (Neary, 1990; Northcutt and Ronan, 1992) or hippocampal formation in mammals (Swanson and Cowan, 1979). However, as the telencephalic hemispheres of ray-finned fishes develops by
eversion and not evagination as in other vertebrate classes,
recognition of homologous telencephalic cell populations
between teleosts and other vertebrates is impaired (see
Nieuwenhuys and Meek, 1990; Braford, 1995; Northcutt,
1995, for recent reviews). Although the telencephalic cell
groups afferent to the Vv (Vid, nT, Dpm) do not readily
correspond to the proposed topological homologue in teleost fishes to the medial pallium, the dorsolateral telencephalon (Northcutt, 1995), further examination of the
connections of these regions will be necessary to fully
assess their evolutionary relationship to the medial pallium.
The most striking similarity between the teleost Vv and
the septal nuclei of other vertebrates is the presence of
connections with cell groups associated with the MFB.
Like the septal nuclei of other vertebrates, the Vv is
heavily connected with the preoptic area and hypotha-
BASAL FOREBRAIN CONNECTIONS IN GYMNOTIFORM FISH
lamic nuclei (teleost fish: Shiga et al. [1985a, 1985b]; Sloan
[1989]; this study; amphibians: Neary and Northcutt
[1990]; Neary [1995]; reptiles: Bruce and Neary [1995a,
1995b]; birds: Berk and Butler [1981]; Székely et al.
[1994]; mammals: e.g., Swanson and Cowan [1979]). Moreover, there appear to be functional relationships in MFB
cell groups. In a related gymnotiform fish, Sternopygus
macrurus, the basal forebrain, preoptic area, and hypothalamic nuclei are immunoreactive for sex steroid receptors
(Gustavson et al., 1994). Therefore, similar to what has
been characterized in the rat (Cottingham and Pfaff,
1986), there appears to be a rich interconnectivity of MFB
cell groups that are target regions for steroid action in
gymnotiforms. Indeed among all classes of vertebrates,
hormone-targeted cell groups appear to be the basal
forebrain and preoptic nuclei in close association with
gonadotrophin-releasing hormone (GnRH) systems (Demski, 1984).
Cell groups that are connected with the Vv also have
neurosecretory cells (PPa, PPp, Hl, Hc, Hv, nLTa), and
have both cell bodies and fibers that are richly endowed
with neuropeptides and monoamines (Johnston and Maler,
1992). Given the extensive anatomical overlap in MFB cell
groups associated with neuroendocrine function, reproductive behavior, and electrocommunication, it will be of
interest to determine their functional relationships and
whether these might be mediated by transmitter-specific
pathways.
ACKNOWLEDGMENTS
I thank Dr. R.G. Northcutt for engaging discussions and
for providing the calretinin antibody used in these studies.
Dr. T.H. Bullock, P. Holmes, Dr. C.H. Keller, Dr. R.G.
Northcutt, and two anonymous reviewers provided helpful
comments on this manuscript. Grace Kennedy and Andrea
Thör provided excellent histological assistance. Additional
thanks to Dr. T.H. Bullock, Dr. J.B. Graham, Dr. R.R.
Hessler, the SIO Director’s Office, the NIH, the NIMH, and
the NSF for allowing the Heiligenberg lab to remain open
for the completion of this work. This work was supported
by NIMH grant R37 MH26149-18, NINCDS grant RO1
NS22244-08, and NSF BNS-9106705 to Dr. W. Heiligenberg.
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