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Afferent and efferent connections of the diencephalic prepacemaker nucleus in the weakly electric fish Eigenmannia virescens interactions between the electromotor system and the neuroendocrine axis

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Afferent and Efferent Connections of the
Diencephalic Prepacemaker Nucleus in
the Weakly Electric Fish, Eigenmannia
virescens: Interactions Between the
Electromotor System and the
Neuroendocrine Axis
The Neurobiology Unit, Scripps Institution of Oceanography, University of California,
San Diego, La Jolla, CA 92093-0201
The afferent and efferent connections of the gymnotiform central posterior nucleus of the
dorsal thalamus and prepacemaker nucleus (CP/PPn) were examined by retrograde and
anterograde transport of the small molecular weight tracer, Neurobiotin. The CP/PPn was
identified by physiological assay and received a local iontophoretic injection of Neurobiotin.
Retrogradely labeled somata were observed in the ventral telencephalon, hypothalamus, and
the pretectal nucleus electrosensorius. Anterogradely labeled fibers were traced from the
CP/PPn to the ventral telencephalon, the hypothalamus, the neuropil immediately adjacent to
the most rostral subdivision of the nucleus electrosensorius, the optic tectum, and the
pacemaker nucleus. Retrograde transport of tracer following injections into the ventral
telencephalon, preoptic area, lateral hypothalamus, tectum, and pacemaker nucleus confirmed these efferent targets. A rostromedial subarea of the CP/PPn can be identified that
projects to basal forebrain regions and to a lateral region of the CP/PPn that contains afferents
to the pacemaker.
Many of the targets, which are connected with the CP/PPn, have been linked to
reproductive behavior or neuroendocrine control in other fishes. A comparative analysis
reveals that the efferent pathways of the CP/PPn appear similar and may be homologous to
efferent pathways of some components of the auditory thalamus among tetrapods. J. Comp.
Neurol. 383:18–41, 1997. r 1997 Wiley-Liss, Inc.
Indexing terms: communication; dorsal thalamus; forebrain; pacemaker; electroreception
The glass knifefish, Eigenmannia virescens, generates a
weak (ca. 10 µV/cm) quasi-sinusoidal electric field in the
water by means of an electric organ, located in its tail. The
electric organ, which is derived from muscle tissue, is
innervated by spinal motoneurons, termed electromotoneurons (see Bass, 1986, for review). Generally, the frequency
of the electric organ discharge (EOD) is fixed, driven by the
medullary pacemaker nucleus. Under certain contexts,
such as the jamming avoidance response (JAR) or novelty
response, the fish can raise or lower its EOD frequency (see
Heiligenberg, 1991, for review). The fish may also produce
interruptions in the EOD during courtship or agonistic
encounters (Hopkins, 1974). These interruptions in
E. virescens are termed chirps (Hagedorn and Heiligenberg, 1985). Both gradual rises in frequency and chirps in
E. virescens are controlled by a nucleus situated in the
thalamus. This bilaterally situated nucleus projects to the
midline pacemaker nucleus and is termed the prepacemaker nucleus (PPn; Heiligenberg et al., 1981; Kawasaki
and Heiligenberg, 1988; Kawasaki et al., 1988; Zupanc and
Heiligenberg, 1989, 1992).
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: Calvin J.H. Wong, The Neurobiology Unit, Scripps
Institution of Oceanography, University of California, San Diego, La Jolla,
CA 92093-0201. E-mail:
Received 20 September 1996; Revised 17 December 1996; Accepted 7
January 1997
There appear to be at least two anatomical and functional subdivisions to the PPn: a lateral magnocellular
region and a medial parvocellular region. Stimulation of
the multipolar cells of the lateral region elicits chirps and
is therefore designated the chirp subdivision (PPn-C).
Stimulation of the ovoid cells of the medial subdivision
elicits gradual rises in EOD frequency and is therefore
designated the gradual rise subdivision (PPn-G). Medially
adjacent to the PPn-G is the central posterior nucleus of
the dorsal thalamus (CP). As the cells of the CP and the
cells of the PPn-G cannot be distinguished from each other,
by using cytoarchitectonic, immunohistochemical, or ultrastructural criteria, the term CP/PPn was developed to
encompass both those cell populations within the region
that specifically project to the pacemaker nucleus (i.e., the
PPn) and those that do not (Zupanc and Zupanc, 1992;
Zupanc and Heiligenberg, 1992). The only other known
input to the pacemaker nucleus is a mesencephalic cell
group, the sublemniscal prepacemaker nucleus (SPPn;
Keller et al., 1991). This nucleus is responsible for gradual
reductions in EOD frequency in E. virescens (Metzner,
1993). In other gymnotiforms, the SPPn appears to control
a variety of other electrocommunicatory behaviors (Kawasaki and Heiligenberg, 1989; Keller et al., 1991; Spiro,
1994; Heiligenberg et al., 1996).
One cell group that projects to the CP/PPn is the
pretectal complex of the nucleus electrosensorius (nE;
Bastian and Yuthas, 1984; Keller et al., 1990). The nE
pretectal nucleus A
anterior commissure
anterior lateral line ganglion
anterior thalamic nucleus
biotinylated dextran amine
ansulate commissure
central nucleus of the inferior lobe
central posterior nucleus (thalamus)
complex of the central posterior nucleus (thalamus) and the
prepacemaker nucleus
tectal commissure
central division of the dorsal telencephalon
dorsal division of the dorsal telencephalon
lateral diffuse nucleus of the inferior lobe
lateral division of the dorsal telencephalon
dorsolateral telencephalon, dorsal subdivision
dorsolateral telencephalon, posterior subdivision
dorsolateral thalamic nucleus
dorsolateral telencephalon, ventral subdivision
medial division of the dorsal telencephalon
dorsomedial telencephalon, subdivision 2, caudal
dorsomedial telencephalon, subdivision 2, dorsal
dorsomedial telencephalon, subdivision 2, rostral
dorsomedial telencephalon, subdivision 2, ventral
dorsomedial thalamic nucleus
posterior division of the dorsal telencephalon
dorsal posterior nucleus (thalamus)
dorsal tegemental nucleus
decussation of the tractus praeminentialis-cerebellaris
caudal enteropeduncular nucleus
eminentia granularis, pars medialis
eminentia granularis, pars posterior
electrosensory lateral line lobe
efferent octavolateral nucleus
electric organ discharge
rostral enteropeduncular nucleus
torus semicircularis efferents
fasciculus retroflexus
anterior hypothalamic nucleus
caudal hypothalamic nucleus
dorsal hypothalamic nucleus
lateral hypothalamic nucleus
ventral hypothalamic nucleus
interopeduncular nucleus
jamming avoidance response
lateral forebrain bundle
lateral lemniscus
Mauthner cell axons
medial forebrain bundle
magnocellular tegmental nucleus
medial longitudinal fasciculus
medial olfactory bulb
anterior periventricular nucleus
nucleus electrosensorius
nucleus electrosensorius, ‘‘up’’ subdivision
nucleus electrosensorius, ‘‘down’’ subdivision
acousticolateralis region of the nucleus electrosensorius
beat subdivision of the nucleus electrosensorius
isthmic nucleus
nucleus of the lateral valvula
nucleus medialis
posterior periventricular nucleus
lateral nucleus of the lateral recess
nucleus taenia
optic commissure
pacemaker cell
posterior commissure
periglomerular nucleus
preglomerular complex
preglomerular complex, lateral subdivision
preglomerular complex, medial subdivision
pacemaker nucleus
postoptic commissure
anterior periventricular preoptic nucleus
prepacemaker nucleus
prepacemaker nucleus, ‘‘chirp’’ subdivision
prepacemaker nucleus, ‘‘gradual rise’’ subdivision
posterior periventricular preoptic nucleus
pretectal nucleus
nucleus prethalamicus
lateral pretectal nucleus
periventricular pretectal nucleus
red nucleus
central raphé nucleus
dorsal raphé nucleus
reticular formation
suprachiasmatic nucleus
sublemniscal prepacemaker nucleus
anterior tuberal nucleus
anterior tuberal nucleus, dorsal subdivision
anterior tuberal nucleus, ventral subdivision
optic tectum
torus longitudinalis
dorsomedial optic tract
posterior tuberal nucleus
periventricular nucleus of the posterior tuberculum
torus semicircularis, dorsal subdivision
torus semicircularis, ventral subdivision
ventral telencephalon, central subdivision
valvula of the cerebellum, medial subdivision
ventral telencephalon, dorsal subdivision
ventral telencephalon, intermediate subdivision
ventral telencephalon, lateral subdivision
ventrolateral thalamic nucleus
ventromedial thalamic nucleus
ventral telencephalon, posterior subdivision
valvular peduncle
ventral telencephalon, supracommisural subdivision
ventral dendritic territory
ventral telencephalon, ventral subdivision
ventral telencephalon, dorsal aspect of the ventral subdivision
ventral telencephalon, ventral aspect of the ventral subdivision
receives ascending electrosensory, mechanosensory lateral
line, and octaval information from the midbrain torus
semicircularis (Carr et al., 1981; Keller et al., 1990), the
homologue of the inferior colliculus. Previous studies have
shown that some neurons in the nE, which appear to fire
preferentially to playbacks of chirps, project to the hypothalamus (Heiligenberg et al., 1991). It has been suggested
that this might provide a substrate for conspecific sensory
signals to influence the motivational and reproductive
state of the animal.
Further support for the presence of interactions between
the electrosensory/motor system and the neuroendocrine
system is suggested by immunohistochemical studies in a
closely related gymnotiform, Apteronotus leptorhynchus.
These studies demonstrate that CP/PPn receives monoaminergic and peptide-ergic innervation which may originate from the hypothalamus (see Johnston and Maler,
1992; Zupanc and Maler, 1997, for review). Of particular
interest is the presence of a sexual dimorphism in substance-P innervation of the CP/PPn (Weld and Maler,
1992). In addition, the extent of substance-P innervation is
correlated with propensity to chirp in testosteroneimplanted female fish (Dulka and Maler, 1994; Dulka et
al., 1995). Other evidence for a functional role of the
CP/PPn in reproductive behavior is the presence of androgen receptors in cells of CP/PPn as well as the nE. These
results suggest that the cells that detect or produce
electrocommunicatory behaviors may be subject to hormonal influence (Zakon, 1993; Gustavson et al., 1994).
Indeed, the PPn-C cells in E. virescens show seasonal
variation in their dendritic structure (Zupanc and Heiligenberg, 1989).
The CP/PPn is a key link of the electrocommunicatory
circuit, residing at the interface of ascending electrosensory information and the eventual transformation of this
information into modulations of the EOD. In spite of this,
only preliminary mapping studies of the connections of
CP/PPn have been published (Keller et al., 1990; Stroh and
Zupanc, 1995). This study was undertaken to further
clarify the organization of this sensory-motor interface by
mapping the afferent and efferent microcircuitry of CP/
PPn. Preliminary results have been published previously
in abstract form (Wong, 1995a,b).
Adult fish (15–20 cm) of both sexes of E. virescens were
used. Injections into the CP/PPn in other gymnotiforms, A.
leptorhynchus and Hypopomus pinnicaudatus revealed
generally similar results, though I will restrict the results
to E. virescens. Some species-specific differences in connectivity were observed between A. leptorhynchus and E.
virescens, particularly with respect to the SPPn, and these
have been presented elsewhere (Heiligenberg et al., 1996).
Fish were obtained from commercial suppliers (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 a
diet of live blackworms. Animal care, anesthesia, surgery,
and euthanasia were carried out in compliance with
guidelines set forth by the Animal Subjects Committee for
the University of California, San Diego.
Anesthesia consisted of placing the fish in a 1:15,000
solution of tricaine methane sulfonate (MS-222; Sigma
Chemical Co., St. Louis, MO) until somatic reactions
stopped. This was followed by immobilization with an
intramuscular injection of 2 µl of 2 mg/ml gallamine
triethiodide (Sigma Chemical Co., St. Louis, MO) in saline.
The fish was then placed in a fish holder with aerated
water perfusing the gills. The surgical site was prepared
with topical application of 20 mg/ml lidocaine, and the
skull was opened to expose the rostral pole of the brain.
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 PPn was localized by physiological assay. A
single-barrel glass micropipette was filled with L-glutamate (0.1 M, pH 8.0), which was iontophoresed with
negatively biased current (100 nA). L-glutamate selectively stimulates cell bodies and dendrites but not fibers of
passage. As the electrode was advanced, the PPn could be
readily identified by modulations in the EOD. Once the
depth and location of the PPn was ascertained (approx.
1,500–2,000 µm in depth), 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 Laboratories, Burlingame, CA). 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 successful reidentification of the site, the tracer
was iontophoresed with positive DC current (ca. 2–5 µA)
for 20 minutes to 1 hour. Following iontophoresis of the
tracer, the electrode was left in place for 5 to 10 minutes,
and then slowly withdrawn to avoid leakage. The hole in
the skull was patched with gelfoam, and the site was
sealed with Vetbond (3M). Survival times were 8 to 30
hours at 27°C for retrograde and anterograde transport of
tracer. Although Neurobiotin was the primary tracer used
in these studies, biotinylated dextran amines (BDA; Molecular Probes, Eugene, OR) were also used (Veenman et
al., 1992). One animal received an iontophoretic injection
of 3,000 molecular weight (3 kDa) BDA. Four animals
received pressure injections of 10 kDa BDA. These other
cases revealed qualitatively similar results, although the
injection sites tended to be larger.
Fish were euthanised in MS-222 and perfused transcardially with 0.9% NaCl followed by fixative for half an hour
(approximately 30 ml of fixative). The fixative was composed of 4% paraformaldehyde and 0.25% glutaraldehyde
in 0.1 M phosphate buffer (pH 7.4). Brains were then
removed and postfixed overnight for tissue processing the
next day.
Tissue processing
Free floating Vibratome sections were processed by
using the avidin-biotin method (ABC Standard Elite Kit;
Vector Laboratories, Burlingame, CA) with a nickel intensified peroxidase-3,3’ diaminobenzidine (DAB) reaction.
Brain sections were processed according to a protocol
modified from previous procedures (Kita and Armstrong,
1991; Lapper and Bolam, 1991; Vaney, 1991; Huang et al.,
1992). The following solutions were used: phosphate buffer
saline (PBS; 0.02 M phosphatebuffer, pH 7.6 and 0.9%
NaCl), 0.1% Triton X-100 in PBS (PBST), and Tris buffer
(TB; 0.1 M Tris buffer, pH 7.2). Vibratome sections were cut
at 50 µm thickness into PBS. They were then ‘‘prebleached’’ by soaking for 10 minutes in 0.5% H2O2 in PBS to
inhibit endogenous peroxidases in the tissue.
By using a Vectastain ABC kit (Vector Laboratories), an
ABC solution was prepared (4 drops A 1 4 drops B in 12 ml
PBST) and stored for 30 minutes. Following prebleaching,
sections were washed three times for 10 minutes in PBS
and incubated in the ABC solution in small, covered dishes
at 4°C for 4–12 hours. Sections were then washed three
times for 10 minutes in PBS and once for 10 minutes in TB,
and then processed by the DAB procedure. Sections were
presoaked for 15 minutes in a solution of TB, 0.064% nickel
ammonium sulfate, and 0.04% DAB (Sigma Chemical Co.,
St. Louis, MO). Following 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. Sections were counterstained with neutral
red (Sigma Chemical Co., St. Louis, MO) and coverslipped
with Permount (Fisher Scientific, Rockville, MD).
Sections were analyzed and compared with reference
cresyl violet and Klüver-Berrera stained paraffin series of
E. virescens as well as the brain atlas of A. leptorhynchus
(Maler et al., 1991), which uses as its basis the nomenclature of a variety of investigators. Generally, the nomenclature of the A. leptorhynchus atlas has been adopted for E.
virescens. Chartings of representative cases were made by
using a camera lucida. Photomicrographs were taken by
using TMAX-100 black and white film (Kodak, Rochester,
NY) with a Wratten 44 filter to enhance the counterstain.
Methodological considerations
Neurobiotin, when iontophoresed, appeared to be taken
up very locally by neurons, with little if any glial label
unless there was ventricular spread. In such instances, a
non-specific gray background reaction product developed
with labeled radial glia. Owing to the low molecular weight
of the tracer, any excess that was not immediately taken
up by cells or terminals in the vicinity of the injection site
appeared to be metabolized or washed out. Fortunately,
the center of the injection was relatively easy to assess, as
there was much heavier cell labeling at the injection site as
well as local tissue damage arising from the iontophoretic
Due to the sensitivity of the technique, anterograde
label of axon collaterals from retrogradely labeled cell
bodies would cause confusion, by suggesting false efferent
targets of the CP/PPn. In cases in which it was not possible
to trace fibers directly to the CP/PPn due to labeling of
such collaterals, reciprocal injections were placed to determine the site of originating cell bodies.
Many connections observed appeared to be reciprocal
with both labeled fibers and terminals. Such patterns of
connections might be explained by transneuronal transport of tracer. There have been some reports that Neurobiotin is transported transneuronally, particularly in cases
where neurons are electrotonically coupled (Vaney, 1991;
Huang et al., 1992). Controls for transneuronal transport
were performed three ways. First, preliminary experiments using BDA, which is not transported transneuronally, retrogradely labeled the same cell groups as Neurobiotin. As BDA injections tended to be larger than
Neurobiotin injections and the rate of transport of BDA
was much slower than that of Neurobiotin, Neurobiotin
was preferable as a tracer in E. virescens.
Second, Neurobiotin injections were made into structures whose connectivity had been extensively documented by previous investigators (i.e., pacemaker, tectum,
torus semicircularis, and cerebellum). Transneuronal transport was observed following injections into lamina VI of
the dorsal torus and following injections into the auditory
torus. With the injections made into lamina VI, retrograde
transport through electrotonic gap junctions extended
from the spherical cells afferent to layer VI, to the cells of
the anterior lateral line ganglion, which electrotonically
synapse onto spherical cells (Carr et al., 1986; Mathieson
et al., 1987). Additionally, massive injections of Neurobiotin into the auditory torus revealed transneuronal transport to fibers that could be traced to the eighth nerve root.
It is uncertain if there is electrotonic coupling in the
central auditory pathways of teleost fishes. Otherwise,
injections into the tectum, torus, and pacemaker nucleus
revealed no connections other than those established by
previous investigators using other tracers.
Finally, in a few fish, longer survival times were used
(.30 hours). In these cases, the extent of label was
actually reduced. This decrease in label is probably due to
metabolism of Neurobiotin (Kita and Armstrong, 1991).
In the fish that received injections of Neurobiotin into
CP/PPn, various iontophoretic protocols were used. Electrode resistance, total injection time, and magnitude of the
injection current had the greatest effects on the discreteness of the injection site. The protocol that was most
commonly used and which yielded discrete injections was a
12 µA square wave pulse, 10 seconds on /5 seconds off for
45 minutes.
Although iontophoretic injections with small diameter
pipette tips (,10 µm) yielded discrete injections (,100 µm
in diameter), uptake by damaged fibers of passage as of
concern due to the small size and complexity of the teleost
diencephalon. To further account for fibers of passage,
hypotheses of connectivity were tested by placing injections reciprocally, and by comparisons of labeled fiber
tracts from over 40 additional injections into other structures from other experiments.
Cytoarchitecture of the CP/PPn
A prominent nuclear complex in the dorsal thalamus of
E. virescens, termed the CP/PPn, can be identified adjacent
to a sulcus in the third ventricle. It is most simply
recognized as a band of cells appearing to extend in a
ventrolateral direction.
Rostrally (Fig. 1A), the CP/PPn is sandwiched between
the third ventricle and the medioventral edge of the
horseshoe-shaped anterior thalamic nucleus (ATh). The
CP/PPn caps the dorsal edge of the ventromedial thalamic
nucleus. At slightly more caudal levels, CP/PPn displaces
the ATh ventrolaterally and forms several lamina parallel
to the ventricular surface (Fig. 1C). At these more caudal
levels, the rostral pole of the periventricular nucleus of the
posterior tuberculum (TPP) flanks the ventral aspect of
the CP/PPn. In addition, it is clear that at this level, the
CP/PPn possesses slightly larger and more diffusely scattered cells along its ventrolateral wing. Further caudally,
the lamina of cell bodies in the CP/PPn appear to fuse, with
the cells appearing as a continuous stream with somata
oriented perpendicular to the ventricular surface (Fig. 1E).
Cells of the CP/PPn at this transverse level are typically
Fig. 1. Rostral to caudal series of photomicrographs through the
diencephalon of E. virescens from a normal reference series (left-hand
column of each page) and a typical injection site (right-hand column)
centered on and generally confined to the complex of the central
posterior nucleus/ prepacemaker nucleus (CP/PPn). A,C,E,G: Rostral
to caudal reference series of transverse sections through the dorsal
thalamus of E. virescens (15 µm paraffin sections, cresyl-violet counterstain). B,D,F,H: Rostral to caudal Vibratome series of injection site in
a single individual corresponding to the same transverse level as the
micrographs to the left (50 µm Vibratome sections, neutral red
counterstain). Center of injection is in F. Alternate sections are shown.
See List of Abbreviations. Scale bars 5 200 µm.
Figure 1
clustered with a cell free boundary separating the smaller,
medial cells from the larger, lateral cells. At the most
caudal extent of the CP/PPn (Fig. 1G), the dorsal posterior
nucleus of the thalamus (DPn) displaces the CP/PPn,
except for the large multipolar cells, which can be observed
for approximately 100 µm caudal to the center of the
Analysis of injection site
Twenty E. virescens received an injection centered on the
CP/PPn. Of these, four animals received a pressure injection of 10 kDa biotinylated dextran amine (BDA). One
animal received an iontophoretic injection of 3 kDa BDA.
The BDA pressure injections tended to be much larger,
encompassing the entire dorsal thalamus. In the remaining 15 cases, iontophoresis of Neurobiotin was used. Of
these, one injection extended rostrally into the ATh and
one injection was centered more ventrally, encroaching on
the TPP and the ventral thalamus. The rest generally
appeared to be confined to the CP/PPn. In two cases, the
injections appeared to be confined to the rostral, medial
portion of the CP/PPn. In these cases, there were no anterogradely labeled terminals within the pacemaker nucleus.
A series of photomicrographs of a confined, laterally
centered injection is presented in Figure 1. From this case,
although the cells right at the center of the injection are
heavily labeled (Fig. 1F), cells located in nuclei which lie
adjacent to the injection site are only sparsely labeled
(ATh, TPP, and DPn). Subsequent uptake from more
medially situated cells delineates the boundary of the
CP/PPn and is most evident at the rostral-most level
where the CP/PPn envelops the ventromedial edge of the
ATh (Fig. 1B). A few cells within the caudal aspect of the
ventrolateral thalamic nucleus (VLTh) showed uptake of
tracer (Fig. 1D) and it is unclear as to whether this is due
to interruption of fibers of passage or represents an actual
afferent projection.
A rostral to caudal charting of a global injection of
Neurobiotin (200 µm diameter) centered on, but not confined to the CP/PPn, is presented in Figure 2. In this case,
the injection was centered in the caudal aspect of the
CP/PPn (Fig. 2K) yet encroached extensively onto rostral
and ventral areas. This injection may have interrupted
fibers originating from the TPP and the ventral thalamus,
as revealed by extensive cell labeling in these nuclei. The
number of labeled somata in these nuclei was considerably
reduced following more restricted injections into the CP/
PPn (e.g., Fig. 1).
Label in the habenula (H), anterior periventricular
nucleus (nAPv), and anterior hypothalamic nucleus (Ha),
which was observed in the global injection of Figure 2, was
not observed with more confined injections. All other labels
charted in Figure 2 were present with injections that were
restricted to the CP/PPn. This suggests that this case
represents the total extent of connections of this nucleus.
There were three exceptions. The optic tectum was observed to be very sparsely labeled in this case. Labeled
somata within the dorsorostral region of the anterior
tuberal nucleus (TA) and within the beat-sensitive subdivision of the nucleus electrosensorius (nEb) were observed
only following medially centered injections but were not
observed in this case.
Afferents to the CP/PPn
Following injections into the CP/PPn, retrogradely labeled cells were most prominent along the neuraxis in cell
groups associated with the medial forebrain bundle. Typically, these areas were reciprocally connected with the
CP/PPn and contained both retrogradely labeled cell bodies and anterogradely labeled terminal fields (see Efferents from the CP/PPn below). These areas include the
basal forebrain, the anterior preoptic area, and some
nuclei in the hypothalamus. Other diencephalic cell groups
projecting to the CP/PPn include the nucleus of the
posterior tuberculum and some subdivisions of the pretectal nucleus electrosensorius. A few retrogradely labeled
cells were also observed in the nucleus of the lateral
valvula, the dorsal and central raphe nuclei, and occasionally within the reticular formation. No projections were
observed from the pallium of the telencephalon, the torus
semicircularis, or the cerebellum.
Ventral telencephalon. The telencephalon of rayfinned fishes is divided into a dorsal part and ventral part
(Nieuwenhuys, 1963; Northcutt and Braford, 1980; Northcutt and Davis, 1983; Braford, 1995, for reviews). The
dorsal part comprises the pallium, whereas the ventral
part comprises the subpallium. The most rostral cell group
projecting to the CP/PPn was observed in a continuum
between the ventral nucleus of the ventral telencephalon
(Vv) and the dorsal nucleus of the ventral telencephalon
(Vd). This cigar-shaped cell group contained retrogradely
labeled perikarya that were typically medium-small (8
µm) and round with lateral extending dendrites. These
cells were clustered in a dorsal subdivision of the Vv (Vv-d:
Fig. 3A,B) continuous with the ventral subdivision of the
Vd. Slightly larger, round, scattered cells were retrogradely labeled in the ventral subdivision of the Vv (Vv-v).
Labeled cells were not consistently observed within the
more dorsal laminar region of the Vd. The extent of
retrogradely labeled somata within the Vv and the Vd was
from approximately 50–100 µm caudal to the olfactory
bulb, extending to the caudal pole of the Vv/Vd for a total
rostral to caudal extent of approximately 600 µm. These
retrograde transport data are corroborated by anterograde
transport of tracer following discrete injections placed in
Vv/Vd (unpublished observations).
Preoptic area. At the level of the anterior commissure
lies the anterior periventricular preoptic nucleus (PPa).
Retrogradely labeled cells were consistently observed in
this region following both global and confined injections of
tracer into the CP/PPn. Within the PPa, labeled mediumsized cells (12–15 µm) were observed approximately 20–30
µm from the periventricular zone (Fig. 3C,D). In contrast
to the PPa, which was characterized by consistently labeled cells, the posterior periventricular preoptic nucleus
(PPp) rarely contained retrogradely labeled cells after
confined injections into CP/PPn. As discussed above, cells
found within the anterior hypothalamic nucleus (Ha),
anterior periventricular nucleus (nAPV), and habenula
(H) depicted from this global injection (Fig. 2E–G) were not
consistently observed after confined injections into the
Hypothalamus. The anterior tuberal nucleus (TA) is a
large complex nucleus, lying ventral to the lateral recess
and dorsal to the lateral hypothalamic nucleus (Hl). It is a
particularly prominent nucleus in gymnotiforms (Striedter,
1992) and consists of at least two distinctive cytoarchitectonic subdivisions (Heiligenberg et al., 1991). Retrogradely
labeled somata were observed only within the dorsal
subdivision and generally only from those injections that
were centered on the rostromedial regions of the CP/PPn
(Fig. 4). These bipolar cells that were labeled were slightly
Fig. 2. Rostral to caudal charting of retrogradely labeled cell
bodies (large dots) and anterogradely labeled fibers (short lines) and
terminals (small dots) following an injection centered on but not
confined to the CP/PPn in E. virescens. A-H: Label in the forebrain.
Arrow in F indicates the rostromedial fiber tract within the medial
forebrain bundle. See text for details. Nomenclature based on the atlas
of a related gymnotiform, A. leptorhynchus (Maler et al., 1991). Scale
bar 5 500 µm. I–L: Label in the diencephalon and rostral mesencephalon. The center of the injection was in part K. An asterisk in part L
demarcates the caudal fiber tract innervating the pacemaker nucleus.
See text for details. Scale bar 5 500 µm. M–O: Label in the caudal
mesencephalon (part M,N) and medulla (part O). Asterisk indicates
caudal fiber tract innervating the pacemaker nucleus. See text for
details. See List of Abbreviations. Scale bar 5 500 µm.
Figure 2
ovoid in shape (7.5 3 10 µm). Following more laterally
placed injections, such as the one charted in Figure 2,
retrograde label within the TA was observed to be considerably reduced.
Within the lateral hypothalamic nucleus (Hl), retrogradely labeled cells were observed lying in close apposition to the boundary between the Hl and the ventral
portion of the TA (Figs. 2J,K, 5A). These retrogradely
labeled cell bodies tended to be more rostrally situated
within the Hl over a rostral to caudal extent of approximately 150 µm. There appeared to be at least two cell types
labeled: ovoid cells (5 3 8 µm) and round cells (approximately 7 µm diameter).
Cells were occasionally retrogradely labeled in the ventral hypothalamic nucleus (Hv). However, since injections
into both the overlying optic tectum and the nearby
Figure 2
nucleus subelectrosensorius heavily retrogradely labeled
somata in the Hv, it is unclear whether the Hv truly
projects to the CP/PPn or if the observed labeling represents leakage of tracer into adjoining areas.
With larger injections of tracer into the CP/PPn, cells
were labeled within the vicinity of the lateral nucleus of
the lateral recess (nRLl; Fig. 5B). Retrograde label within
this nucleus occurred only in a few cases in which the
injection site did not appear to be confined to the CP/PPn.
Further caudally, retrogradely labeled cell bodies were
found within the caudal hypothalamic nucleus (Hc), situated primarily along the lateral edge of the nucleus
abutting the basal hypothalamic tract (tBH). These cells in
the Hc (Fig. 5C) were generally slightly ovoid in shape (ca.
8 3 10 µm) and adendritic.
The central nucleus of the inferior lobe (CE) lies immediately caudal to the lateral recess. A group of medium-large,
slightly ovoid cells were observed to be retrogradely labeled within the CE (Fig. 5D). These cells are 10–15 µm
across with extensive dendritic arbors that generally
remain confined to the CE.
Nucleus electrosensorius. Nucleus electrosensorius
(nE) is a pretectal nucleus extending from the nucleus
prethalamicus caudal to the interstitial reticular area,
consisting of several subdivisions (Keller et al., 1990;
Heiligenberg et al., 1991).
The most rostrally situated subdivision is the acoustic
and lateral line region of the nE (nEAR), which receives
input from the ventral subdivision of the torus semicircularis (Carr et al., 1981; Keller et al., 1990). Within the
nEAR and in the neuropil medial to it, a few labeled cells
were observed to be retrogradely labeled.
Labeled cells were also observed within the beatsensitive subdivision of the nE (nEb). These cells project
into the vicinity of the CP/PPn by means of a tight fascicle
of fibers which course immediately dorsal to the preglomerular complex. These relatively large multipolar cells
(Fig. 6A) of the nEb (10–15 µm) have dendrites that ramify
extensively within the confines of the nucleus. Cells within
this region were retrogradely labeled from medially placed
The most caudal subdivision of the nucleus electrosensorius is termed the nE-up (nE>) as stimulation of this region
with L-glutamate can elicit gradual rises in EOD frequency. Small adendritic ovoid cells (5 3 10 µm) in the nE>
were observed to be retrogradely labeled following CP/PPn
injections (Fig. 6B). The axons from nE> cells appeared to
enter the CP/PPn by a ventromedially directed band of
Other afferents to the CP/PPn. Retrogradely labeled
cells were observed in the nucleus of the posterior tuberculum (TP). These small ovoid cells (5 3 10 µm) were
Fig. 3. Label in the forebrain following an injection into CP/PPn. A:
Retrogradely labeled cells and anterogradely labeled fibers and terminals within the ventral telencephalon ipsilateral to a large injection of
Neurobiotin into the CP/PPn. Cytoarchitectonic boundaries indicated
on the contralateral side. B: High-power micrograph of labeled cells
within the dorsal subregion of the ventral nucleus of the ventral
telencephalon (Vv-d) following a more confined injection into the
CP/PPn. C: Labeled fibers and cells within the forebrain at the level of
the anterior commissure following an injection of 3 kDa biotinylated
dextran amine (BDA) into CP/PPn. D: High-power photomicrograph of
neurons labeled in the anterior periventricular preoptic nucleus (PPa).
Same section as in C. E,F: Labeled fibers within the medial forebrain
bundle arising from an injection of 3 kDa BDA centered on but not
confined to the CP/PPn. Note that labeled cells in the anterior hypothalamus (Ha) in part E were not observed with smaller injections
although labeled fibers were. E is 100 µm rostral to F. Scale bars 5 100
µm in A,C, 20 µm in B, D, 200 µm in E,F.
Fig. 4. Photomicrograph of an injection into the rostromedial
subdivision of the CP/PPn with labeled cells and terminals within the
lateral nucleus of the ventral thalamus (VLTh) and the dorsal
subdivision of the anterior tuberal nucleus (TAd). Scale bar 5 100 µm.
distributed bilaterally over the entire rostrocaudal extent
of the TP.
Retrogradely labeled cells were also found in the dorsal
and central raphe nuclei. Labeled cells in the dorsal raphe
were generally more consistently observed than in the
central raphe. As serotonergic fiber bundles are extensive
in the diencephalon in gymnotiforms (Johnston et al.,
Fig. 5. Label cells and fibers in the hypothalamus. A: Labeled cells
and fibers in the lateral hypothalamus (Hl). B: Heavily labeled fibers
and cells within the lateral nucleus of the lateral recess (nRLl) from a
global injection into the CP/PPn. Uncounterstained section. C: Retrogradely labeled neurons and anterogradely labeled fibers in the caudal
hypothalamus. D: Retrogradely labeled neurons in the central nucleus
of the inferior lobe. Differential interference contrast optics. See List of
Abbreviations. Scale bar 5 100 µm in A–C, 50 µm in D.
Fig. 6. Retrograde label in the nucleus of the electrosensorius (nE). A: Retrogradely labeled cells in the
beat sensitive subdivision (nEb). B: Retrogradely labeled neurons in the ‘‘up’’ subdivision of the nucleus
electrosensorius (nE>). Scale bar 5 20 µm for both A,B.
1990), it is unclear which of these cell groups provides
serotonergic innervation to the CP/PPn.
Following CP/PPn injections, retrogradely labeled cells
were consistently observed within the nucleus of the
lateral valvula (nLV). However, the number of cells within
this very large nucleus are few and may represent uptake
by fibers of passage. Following injections of tracer into the
nLV, extensive retrograde and anterograde label were
observed in the diencephalon, with particularly prominent
terminal fields in nuclei adjacent to the CP/PPn. Only a
few sparsely anterogradely labeled fibers were observed in
the CP/PPn.
Efferents from the CP/PPn
Anterogradely labeled fibers and terminals following
CP/PPn injections are charted with afferents in Figure 2.
However, as Neurobiotin is transported in both retrograde
and anterograde directions, observed labeled fibers and
terminals may be collaterals of retrogradely labeled cell
bodies. Therefore, the description of efferent fibers originating from the CP/PPn will be focused on those that were
corroborated by subsequent placement of reciprocating
injections (see Retrograde transport below). One exception
is the projection into the medial shell of nEAR, which was
particularly prominent and consistently observed with
discrete CP/PPn injections.
Anterograde transport
Ventral telencephalon.
A rostromedial bundle was
observed to emerge from the injection site. This bundle
courses in a paraventricular position within the medial
forebrain bundle, medial to the rostral-most pole of the
dorsal thalamus. A few fibers from this rostromedial
bundle could be traced through the postoptic commissure
to the contralateral medial forebrain bundle. At the transverse level of the suprachiasmatic nucleus, the fiber bundle
courses in a ventrolaterally opening crescent along the
dorsomedial aspect of the diencephalon (Figs. 2F, 3E,F).
The arms of the crescent divide at the telencephalicdiencephalic junction, giving rise to dorsally coursing
fibers that innervate the rostral midline subdivisions of
the ventral telencephalon and ventromedially running
fibers that innervate the preoptic area (Figs. 2A–F, 3).
There was no evidence of any labeled fibers within the
lateral forebrain bundle.
Anterogradely labeled fibers and terminals, partially
coextensive with retrogradely labeled neurons, were found
within the midline nuclei of the ventral telencephalon in a
continuum of the Vd-v, the Vv-d, extending caudally to the
supracommisural nucleus of the ventral telencephalon
(Vs). These anterogradely labeled fibers never extended
into the dorsal portion of the Vd, even after massive
injections into the CP/PPn. Projections from the CP/PPn to
the pallium were not observed.
Injections into the CP/PPn yielded
extensively labeled fibers and terminals within the PPa,
and the PPp. These putative anterogradely labeled terminals were most prominent within the nucleus proper, with
sparser label in the lateral neuropil.
Labeled fibers were seen emanating from the center of
the injection site, entering the rostral segment of the TA,
and innervating primarily the rostral, dorsal region (TAd)
coextensive with the retrogradely labeled cell bodies.
Anterogradely labeled fibers were only occasionally observed in the ventral portion of the TA.
The lateral hypothalamus contained fibers and putative
terminals from CP/PPn injections. The fibers innervating
the lateral hypothalamic nucleus originate from the injection site and arc ventrolateral around the TA, rostral to the
lateral recess. The fiber bundle was observed to turn
medially and innervate the lateral hypothalamus over its
entire rostrocaudal extent (approximately 600 µm). Extensive fiber labeling was also evident within the nRLl (Fig.
5B). More caudally, within Hc and its surround there were
frequently anterogradely labeled fibers with clear en
passant terminals (Fig. 5C). The inferior lobe however
lacked anterogradely labeled fibers except following injections that did not appear to be confined to the CP/PPn.
Nucleus electrosensorius. Following injections into the
CP/PPn, a band of labeled fibers was observed to innervate
the neuropil immediately medial to the nEAR. This region
Fig. 7. A: Fiber and terminal labeling within the neuropil, medial
to the acousticolateralis subdivision of the nucleus electrosensorius
(nEAR) following a confined injection of Neurobiotin into the CP/PPn.
A few cells are also labeled within this region as well. Dashed line
indicates the traditionally recognized medial boundary of the nEAR.
B: High power of photomicrograph A. Scale bars 5 100 µm in A,B.
is relatively cell sparse and is situated between the
pretectal nucleus and the ATh. The anterogradely labeled
fibers appear to exit from the CP/PPn and arc around the
ATh. Labeled fibers and terminals were observed in a
dense plexus, enwrapping the nEAR in a rostromedially
situated shell (Fig. 7).
Pacemaker nucleus. Along the base of the brain, interlaced with the commissural fibers of the decussation of the
praeminential-cerebellar tract, run the fibers afferent to
the pacemaker nucleus. These descending fibers project
primarily ipsilaterally to the level of the pacemaker nucleus
where they appear to coalesce at the midline and sweep
dorsally in a tight fascicle flanked by the medial longitudinal fasciculus. These descending fibers enter the pacemaker nucleus from the ventral edge, procuring terminals
throughout the entire extent of the midline nucleus (Fig.
8A). The pacemaker nucleus consists of two cell types:
smaller, intrinsic pacemaker cells and larger relay cells
whose thick diameter axons project out of the nucleus
down the spinal cord (reviewed in Dye and Meyer, 1986).
Both cell types receive dense innervation from the CP/PPn
with clear terminal punctata enveloping the cells (Fig. 8B).
Other efferents of CP/PPn. In cases where the injection
site was not limited to CP/PPn, the intermediate nucleus
of the ventral telencephalon (Vi) also contained anterogradely labeled fibers. Cases in which there was no tracer
leakage outside of the CP/PPn, the Vi showed only sparse
Labeled fibers were occasionally observed in the optic
tectum following large injections into the CP/PPn. These
labeled fibers in the tectum were generally restricted to
the stratum albumen centrale (see Sas and Maler, 1986 for
details on tectal organization in gymnotiforms).
Anterogradely labeled fibers were also observed in the
vicinity of the SPPn in E. virescens; however their source
could not be traced specifically to the CP/PPn.
Retrograde transport. The relative distribution of
efferent cells of the CP/PPn was investigated by placing
Fig. 8. Anterograde label in the pacemaker nucleus (Pn).
A: Anterograde label in the pacemaker nucleus following a large
injection of BDA in the CP/PPn. A single cell in the reticular formation
adjacent to the pacemaker nucleus is retrogradely labeled (arrow).
B: High-power photomicrograph (oil immersion) of putative anterogradely labeled terminals in the pacemaker nucleus. A pacemaker cell
(P) is shown. Same case as A. See list of Abbreviations. Scale bars 5
200 µm in A, 25 µm in B.
injections into the pacemaker nucleus (n56), ventral telencephalon (n54), preoptic area (n52), hypothalamus (n51),
and optic tectum (n51) for retrograde transport of tracer.
Cell bodies originating from the CP/PPn and vicinity are
charted in Figure 9.
An example of retrogradely labeled neurons, following
an injection into the pacemaker nucleus, is charted in
Figure 9A. Observed labeled cells were situated within the
ventrocaudal regions of the CP/PPn (Fig. 9A). No cells
were ever found at the most rostral pole of the CP/PPn
where the nucleus forms a shell encapsulating the ATh. In
contrast, cell bodies within the CP/PPn that project to the
Vv and the Vd were consistently observed within this
rostral region (Figs. 9B,C, 10A,B) but not within the more
caudolateral region of the nucleus.
Two injections were placed in the preoptic area, with one
centered in the PPa and one centered more caudally at the
boundary of the PPa and the PPp. Figure 9D documents
the more caudally centered injection. The neurons that
were retrogradely labeled within the CP/PPn were indistinguishable in morphology and distribution from those that
project to the ventral telencephalon. This suggests that the
same neurons within the CP/PPn may project to both the
ventral telencephalon and the preoptic area.
Following an injection into the optic tectum retrogradely
labeled cells were observed in the CP/PPn (Fig. 9E). Many
other nuclei in the vicinity of the CP/PPn also project much
more heavily to the optic tectum than does the CP/PPn.
These include the dorsal posterior thalamic nucleus (DPn)
and the periventricular pretectal nucleus (PTPv).
Figure 9F documents an injection centered on but not
confined to the nucleus of the lateral recess and the lateral
hypothalamus. In this case, large, round retrogradely
labeled cells were observed in the CP/PPn (Fig. 10C). The
CP/PPn cells that project to the hypothalamus appear to
overlap extensively with the pacemaker projecting cells.
Intrinsic connections of the CP/PPn
The placement of the injection into the CP/PPn was
determined by iontophoresis of L-glutamate into the CP/
PPn to stimulate the PPn cells and elicit chirps laterally
and gradual rises medially. Following injections that were
centered and appeared confined to the lateral portion of
the CP/PPn, neurons were always observed in medial and
rostral regions of the nucleus. In contrast, following confined, medially centered injections, no cells were observed
in the lateral portion of the nucleus although extensive
anterogradely labeled fibers were present (Fig. 11A). The
location of these fibers and putative terminals were in the
vicinity of the prepacemaker cells, in particular the ventral dendritic territory (VT) of the PPn-C cells (Fig. 11B).
Moreover, there were no terminals observed in the pacemaker, suggesting that a subset of cells within the medial
CP/PPn projects laterally to the pacemaker-projecting
cells (Fig. 11B).
The present study provides a fine-grained analysis of the
connections of the CP/PPn in E. virescens. This confirms
and extends previous connectional (Heiligenberg et al.,
1981; Kawasaki et al., 1988; Keller et al., 1990; Johnston
and Maler, 1992; Stroh and Zupanc, 1995) and immunohistochemical studies on gymnotiform fishes (see Johnston
and Maler, 1992; Zupanc and Maler, 1997, for reviews).
The results of the present study are summarized in
Figures 12 and 13.
The dominant sensory input to the CP/PPn appears to be
electrosensory and possibly mechanosensory lateral line
from the nE, which relays ascending information from the
torus semicircularis (Carr et al., 1981; Keller et al., 1990).
Three subdivisions of the nE project to the CP/PPn: the
nE>, the nEb, and the nEAR, although this latter pathway
appears to be relatively weak. Cells of the CP/PPn project
through the medial forebrain bundle to the midline nuclei
of the ventral telencephalon (Vv and Vd) and to the
preoptic area (PPa, and possibly Ha and PPp). Other
intrinsic diencephalic projections are primarily to the
hypothalamus (Hl, Hc, and nRLl). Many of these areas
also project back to the CP/PPn (Vv, Vd, PPa, Hl, and Hc),
suggesting that the CP/PPn may be a critical linkage in
the medial forebrain bundle circuit of gymnotiforms.
The CP/PPn appears to consist of distinctive subregions
with unique inputs and outputs. The rostromedial portion
appears to be the major recipient of input from the forebrain, the nEb, and the TAd as well as a major source of
afferents to the basal forebrain. This rostromedial portion
also projects caudolaterally to the PPn cells, which in turn
constitute the major descending output of this complex.
The projection to the Vs (Fig. 12) was consistently
observed; however, it could not be completely corroborated.
As it was possible to place injections in the Vv/Vd, which
excluded the Vs, afferent innervation to the Vv from the
CP/PPn could be confirmed. In contrast, since it was not
possible to place injections in the Vs that excluded the
Vv/Vd, it remains unclear as to whether the CP/PPn
projects to the Vs.
Thalamic connections in teleosts
The term CP/PPn was developed to encompass both the
CP of the dorsal thalamus, which is a nuclear complex
recognized in other teleosts, and the PPn (Zupanc and
Heiligenberg, 1992). The present cytoarchitectonic description of the CP/PPn is in agreement with previous descriptions of the CP in other closely related teleosts (Braford
and Northcutt, 1983; Striedter, 1990a). Similarly, many of
the connections identified in the present study have also
been described among other teleost fishes. Comparisons
between connections identified in this study and those
identified on previous studies on the thalamus in other
teleosts will be discussed as telencephalic connections,
intrinsic diencephalic connections, and mesencephalic connections.
Telencephalic connections. Projections from the
thalamus to the ventral telencephalon have been reported
among a variety of fishes by both retrograde methods
(Sloan, 1989; Striedter, 1990b, 1991; Shiga et al., 1985;
Murakami et al., 1986) and anterograde methods (Ito et
al., 1986). In the common goldfish (C. auratus) and the
channel catfish (I. punctatus), the cell bodies of origin were
specifically located in the CP (Sloan, 1989; Striedter,
1990b, 1991). In studies on the other teleosts (Shiga et al.,
1985; Murakami et al., 1986), although it is clear that the
thalamus projects to the ventral telencephalon, which
nucleus of the thalamus that actually projects cannot be
readily determined as these investigators used the parcellation scheme of Schnitzlein (1962). This scheme does not
demarcate the CP. Nonetheless, thalamic projections to
the ventral telencephalon are probably shared among
teleost fishes.
In contrast, only a single retrograde transport study
from the telencephalon (Gnathonemus petersii; Wulliman
and Northcutt, 1990) has identified a projection from the
CP to the dorsal telencephalon or pallium. Such a projection could not be identified in E. virescens. It is unclear if
this represents a real species difference, or if the pallial
injection sites in G. petersii encroached onto the subpallium, and thus could account for retrograde label in the CP.
Further work on other fishes, with more discrete injections, will be necessary to ascertain if there is phylogenetic
variation in a projection from the CP to the pallium.
No other study has identified descending projections to
the thalamus from the ventral telencephalon, although
only a single studied employed relatively discrete injections into the thalamus (Ito et al., 1986). Anterograde
transport data supports the presence of these descending
connection in E. virescens (unpublished observations).
Intrinsic diencephalic circuitry. There have been
few studies that specifically examine the intrinsic microcircuitry of the teleost diencephalon (Shiga et al., 1985;
Sloan, 1989; Keller et al., 1990; Wulliman and Northcutt,
1990; Striedter, 1991, 1992; Lamb and Caprio, 1993;
Wulliman and Roth, 1994). Similar to projections to the
ventral telencephalon, thalamic projections to the preoptic
area have also been described (Shiga et al., 1985; Sloan,
1989) and it is likely that thalamic projections to the
preoptic area may also be shared among teleosts.
Projections from the CP to the TA have been described
previously in the channel catfish (Striedter, 1991) and in
E. virescens (Keller et al., 1990). Reciprocal connections
between the CP/PPn and other hypothalamic nuclei had
also been suggested by previous immuohistochemical studies on both E. virescens and A. leptorhynchus (Sas and
Maler, 1991; Zupanc et al., 1991; Weld and Maler, 1992;
Yamamato et al., 1992; Dulka et al., 1995; Richards and
Maler, in press). It is unknown whether these other
hypothalamic nuclei are connected with the thalamus
among non-gymnotiform fishes.
In contrast to a previous in vitro tracing study on A.
leptorhynchus (Striedter, 1992), no connection was observed between the CP/PPn and the preglomerular complex in E. virescens (this study) or A. leptorhynchus
(unpublished observations). As the efferents of the CP/PPn
that project to the lateral hypothalamus clearly pass near
the preglomerular complex, inadvertent labeling might
explain Striedter’s findings. Indeed, in the channel catfish,
a connection between the CP and the preglomerular
complex was not observed (Striedter, 1991, 1992).
Fig. 9. A–F: Retrograde confirmation of efferents of the CP/PPn.
Left hand side indicates center and approximate extent of injections
(black area in left hand transverse section) yielding retrograde labeled
cells (open circles) within the CP/PPn and vicinity. Each dot indicates
a single cell labeled in that section. Every other section is presented
corresponding to approximately the same rostral-caudal levels shown
in Figure 1. See text for details. Scale bars 5 500 µm for injection sites
and 250 µm for chartings of labeled cells in the CP/PPn.
Previous retrograde studies have suggested that the CP
may project to the pituitary in both goldfish (Johnston and
Fryer, 1990) and A. leptorhynchus (Johnston and Maler,
1992). Such a projection was not observed in the present
study, except in a single case: a large injection of BDA to
the CP/PPn. However, even after this large injection, only
a single fiber was observed in the pituitary. The present
study suggests that any direct influence from the CP/PPn
on the pituitary in E. virescens is minor.
Mesencephalic connections. In A. leptorhynchus, a
prominent projection was observed from cells of the CP/
PPn to the SPPn (Heiligenberg et al., 1996). In E. vire-
scens, such a projection could not be readily identified. In
the present study, a few anterogradely labeled fibers were
observed in the vicinity of the SPPn, following injections
into the CP/PPn of E. virescens. However, their source of
origin could not be readily determined as originating from
the CP/PPn. The fiber tract, linking the CP/PPn and the
SPPn, which was clearly observed in A. leptorhynchus, was
not present in E. virescens and hence this may represent a
species-specific difference. A projection was observed from
the CP to the optic tectum in the channel catfish (Striedter,
1990b) and from the CP/PPn in E. virescens (this study).
However, input from the CP/PPn to the optic tectum would
Figure 9
appear to be relatively minor in comparison to projections
originating from other diencephalic nuclei. Conspicuously
absent in E. virescens was a connection between the
CP/PPn and the torus semicircularis. Previous anatomical
(Echteler, 1984; Striedter, 1991) and physiological studies
(Echteler, 1985; Lu and Fay, 1995) on other teleosts have
suggested that there is a direct reciprocal connection
between the midbrain auditory nucleus in the torus semicircularis and the CP. The ascending portion of this
pathway may be homologous to the auditory pathway
through the dorsal thalamus of other vertebrates (see
McCormick, 1992, for a review). Large injections of either
Neurobiotin or BDA into the CP/PPn failed to label cells or
terminals within either the ventral or dorsal subdivisions
of the torus semicircularis, except when there were instances of leakage along the electrode tract into the torus.
Such leakage was accompanied by labeled fibers in the
lateral lemniscus. Similarly, discrete injections of tracer
into either the ventral or dorsal subdivisions of the torus
semicircularis also failed to anterogradely label terminals
within the CP/PPn although other diencephalic fields were
clearly labeled (C.J.H. Wong, unpublished observations).
These data suggest that linkages between the torus semicircularis and the CP/PPn in gymnotiforms are indirect,
mediated through the nE, and are not direct as in other
teleosts. The connections of the medial CP/PPn of E.
virescens are very similar to those of CP of other teleosts.
Based on topology, cytoarchitecture, and connections, these
Fig. 10. A: Photomicrograph of the injection site centered at the
interface of the ventral telencephalon, ventral subdivision (Vv) and
the dorsal nucleus of the ventral telencephalon (Vd), depicted in
Figure 9B. B: Example of retrogradely labeled neurons in the CP/PPn
from the injection in A. C: Example of retrogradely labeled neurons in
the CP/PPn from the injection centered on but not confined to the
lateral nucleus of the lateral recess (shown in Fig. 9F). Scale bars 5
200 µm in A, 100 µm for B,C.
areas are clearly homologous. One of the major efferent
targets of the medial CP/PPn are the PPn cells (this study)
that control EOD modulations for conspecific communication (Kawasaki et al., 1988). Previously, it has been
hypothesized that the dorsal thalamus may subserve
motor circuits for reproductive behaviors in other fishes
(Demski and Dulka, 1986; Zupanc and Heiligenberg, 1992).
In A. leptorhynchus, most of the CP/PPn cells that actually
project to the pacemaker are in fact somatostatinergic
(Stroh and Zupanc, 1995). Examination of both the projection patterns and chemical identity of cells in the dorsal
thalamus of other teleosts may be instructive in determining if the pacemaker-projecting component of the CP/PPn
of gymnotiforms has a homologue in other fishes.
E. virescens, although it is not necessary for the JAR of
A. leptorhynchus (Heiligenberg et al., 1996).
The nEb is also retrogradely labeled from injections into
the CP/PPn confirming previous tract tracing (Keller et al.,
1990) and intracellular labeling (Heiligenberg et al., 1991)
studies. The cells of the nEb project to rostromedial areas
of the CP/PPn. Neurons of the nEb have been recorded
from that respond to beat modulations in electrosensory
input. These would arise from interference of a neighboring fish’s EOD (Keller, 1988; Heiligenberg et al., 1991).
Although the nEb is not necessary for the JAR, it may be
used for recognition and detection of conspecific EOD’s
(Heiligenberg et al., 1991).
The nEAR provides a weak projection to the CP/PPn.
This region of the nE region receives mechanosensory and
ampullary electrosensory input from the ventral torus
semicircularis (Keller et al., 1990; Heiligenberg et al.,
1991; see Metzner and Viete, 1996, for review). The
projection from the nEAR to the CP/PPn would appear to
be relatively minor in comparison to inputs from other nE
subdivisions to the CP/PPn.
The nE may also offer indirect pathways to the CP/PPn.
One such pathway may pass through the anterior tuberal
nucleus of the hypothalamus. One of the major efferent
targets of the nEb is the TAd (Keller et al., 1990; Heiligenberg et al., 1991), which is also reciprocally connected with
Functional considerations
Parallel pathways from the nucleus electrosensorius
to CP/PPn. The present study identifies at least five
parallel pathways which may subsequently relay information from the nE to the CP/PPn (see Fig. 13).
The most prominent subnucleus projecting to the CP/
PPn is from the most caudal subdivision of the nE, the nE>
(Bastian and Yuthas, 1984; Keller, 1988; Keller and Heiligenberg, 1989; Keller et al., 1990). This confirms previous
tract-tracing studies by Keller et al. (1990). This pathway
appears to be necessary for the JAR (Metzner, 1993) of
Fig. 11. A: Anterogradely labeled fibers in the caudal portion of the
CP/PPn arising from a medially and rostrally placed injection (same
case as depicted in Fig. 4, 150 µm caudal to the section in Fig. 4).
B: Retrogradely labeled neurons in the CP/PPn from an injection into
the pacemaker nucleus. Note that in A, there are very few labeled
neurons though extensive fibers and putative terminals in the lateral
region where the prepacemaker neurons typically label. See text for
details. See List of Abbreviations. Scale bar 5 100 µm for A,B.
the CP/PPn (Keller et al., 1990; this study). Further
physiological investigation of the TAd will be necessary to
examine the function of this pathway.
A second indirect pathway may pass through the inferior
lobe. Intracellular recording from cells within the nEAR
has shown that there are combination sensitive units that
fire preferentially to mimics of chirps (Heiligenberg et al.,
1991). Single cell intracellular labeling as well as discrete
extracellular injections have revealed that among the
efferent targets for some of these chirp-sensitive cells is
the central nucleus of the inferior lobe (Keller et al., 1990;
Heiligenberg et al., 1991). As cells within the CE retrogradely label from the CP/PPn, this may represent another
link, relaying sensory chirp information.
In addition to the various ascending pathways to the
CP/PPn from the nE, there may be a feedback pathway
present. The chirp-sensitive cells in the PT/nEAR have
extensive dendrites that appear to extend into the neuropil
medial to the nEAR (Heiligenberg et al., 1991). As the
CP/PPn projects into this area, there might be contact
between the cells in the CP/PPn which generate chirps and
the nEAR cells which detect them.
Links with reproductive centers. The cells of the
CP/PPn are reciprocally connected with cell groups of the
basal forebrain, preoptic area, and hypothalamus. These
regions have been implicated in reproductive behaviors
and the neuroendocrine control of reproduction in teleosts
as well as other vertebrates (see Demski, 1984, for a
review). For example, in goldfish, a direct preopticospinal
pathway that mediates gamete release has been identified
(Demski and Sloan, 1985; Sloan, 1989). Furthermore,
gonadotropin-releasing hormone (GnRH) immunoreactive
somata have been identified in the Vv and the PPa among
a variety of teleosts (Demski, 1984). In addition, many
nuclei that are in receipt of input from CP/PPn (Vv, PPa,
PPp, Hl, and Hc) also have cells that project to the
pituitary (Johnston and Maler, 1992).
Besides receiving input from the CP/PPn, the Vv, Vs and
PPa also receive input from the medial olfactory bulb
(Bass, 1981; Levine and Dethier, 1985; Sas et al., 1993). In
other teleost fishes, there is evidence that the medial
olfactory bulb may encode sex pheromonal information
(see Dulka, 1993 for review). It will of interest to examine
if gymnotiforms utilize chemical cues in addition to electrosensory signals in their reproductive behavior.
In addition to anatomical evidence, both behavioral and
physiological data support a role for many of these regions
in reproductive behaviors. Lesions of the Vs and Vv reduce
reproductive behavior in goldfish (Kyle and Peter, 1982;
Kyle et al., 1982). Similarly, stimulation of the ventral
Fig. 12. Schematic side view of the gymnotiform brain displaying
connections with the CP/PPn; those identified in the present study are
indicated by bold lines. Double asterisks (**) indicates that this
nucleus is an efferent target of the nE. Minor connections (e.g., tectum,
raphé, etc.) are not presented in this summary for clarity. Dashed lines
indicate connections for which there is physiological evidence (sublem-
niscal prepacemaker nucleus, SPPn) or which is suggested (supracommisural nucleus of the ventral telencephalon, Vs) by the presence of
anterogradely labeled fibers and terminals yet which could not be
confirmed by retrograde transport. The arrow within the CP/PPn
demarcates connectional differences between the rostromedial regions
and the caudolateral regions of this nucleus. See text for details.
telencephalon, preoptic area, and hypothalamus in a variety of teleosts can elicit reproductive behaviors, including
gamete release, nest-building, as well as a host of speciesspecific communicatory signals (Demski and Knigge 1971;
Satou et al., 1984; Fine and Perini, 1994; reviewed in
Demski, 1983). Preliminary results suggest that electrical
stimulation of these brain areas in E. virescens elicits EOD
modulations (C.J.H. Wong, unpublished observations),
implicating a role of these regions in electrocommunication.
Comparisons with other vertebrates
Fig. 13. Possible parallel pathways through the diencephalon,
which may relay ascending electrosensory information from the torus,
through the nucleus electrosensorius (thick lines) to the CP/PPn.
Lines in grey are those connections primarily associated with the
medial portion of the CP/PPn. The thick dashed line indicates a
putative feedback connection as the axons originating from the
CP/PPn may be contacting medially directed dendrites of the cells of
the nEAR. Thin dashed line indicates a very weak connection from the
nEAR to the CP/PPn. See List of Abbreviations.
Interestingly, the connections identified for the CP/PPn
appear to be similar to those identified for the proposed
homologue in anuran amphibians: the central thalamic
nucleus (Neary and Northcutt, 1983; Northcutt, 1995).
Unfortunately, as detailed data is lacking to adequately
compare all connections of the CP/PPn with the CP among
non-teleost fishes (see Northcutt, 1995), it is too early to
determine the primitive condition of the octavolateralis
pathways in anamniotes. One may, however, put forward
hypotheses based on comparisons between the CP/PPn of
E. virescens and the central thalamic nucleus of frogs.
These hypotheses will become testable as more data is
gathered among other classes of fishes.
The ascending electrosensory pathway from the CP/PPn
to the telencephalon appears to be similarly organized to
auditory pathways in anuran amphibians. The central
nucleus of frogs, which receives direct input from the
auditory torus, is reciprocally connected with the septum
and projects to the striatum (see Neary, 1990; Northcutt,
1995, for reviews). Although still somewhat contentious,
homologues for telencephalic areas in receipt of input from
the CP/PPn are lateral septum and nucleus accumbens for
the Vv and striatum for the Vd (see Northcutt, 1995, for
review). Also similar to what has been identified in E.
virescens, the hypothalamus and preoptic area may receive
input from the central nucleus in anuran amphibians
(Hall and Feng, 1987; Allison and Wilczynski, 1991; Neary,
1995). Therefore, pending examination of other fishes, it
would appear that connections with septal and striatal
areas of the telencephalon, as well as the preoptic area and
hypothalamus may be primitive for the dorsal thalamic
octavolateralis nucleus in anamniotes.
Owing to the tremendous expansion of the dorsal thalamus between amniotic vertebrates versus anamniotic vertebrates (Butler, 1994), direct comparisons between their
individual thalamic nuclei are complicated. Nonetheless,
it is likely that the central nucleus of anuran amphibians
and hence CP/PPn may be homologized to at least some
portions of the auditory thalamus in amniotes. Projections
to subpallial areas of the telencephalon and the hypothalamus from dorsal thalamic auditory nuclei have also been
described in birds (Durand et al., 1992; Cheng and Zuo,
1994) and mammals (LeDoux et al., 1985; see Frost and
Masterton, 1992, for review). These may be homologous to
the pathways discussed above in frogs and in E. virescens.
All these pathways appear similar in serving reproductive/
autonomic functions (LeDoux et al., 1985; Kawasaki et al.,
1988; Allison, 1992; Wilczynski et al., 1993; Cheng and
Zuo, 1994), suggesting that this might be a primitive
functional feature of dorsal thalamic octavolateralis pathways.
I thank Dr. R.G. Northcutt for helpful guidance and
discussions throughout on this project and for comments
on an earlier draft of this manuscript. Additional thanks to
Dr. T.H. Bullock, Dr. J.B. Graham, the SIO Director’s
Office, the NIH, and the NIMH, for allowing the Heiligenberg lab to remain open for the completion of this work.
Grace Kennedy provided excellent technical assistance.
Dr. L. Maler, Dr. J.C. Prechtl, and Dr. G.K.H. Zupanc
provided helpful discussions during their tenure as visiting scholars to the Heiligenberg lab. I gratefully acknowledge C.B. Braun, Dr. T.H. Bullock, Dr. J.G. Dulka, Dr. C.H.
Keller, Dr. L. Maler, K.T. Moortgat, M.-S. Northcutt, and
two anonymous reviewers for invaluable suggestions on
improvements to this manuscript. This paper is dedicated
to the memory of a great mentor, Dr. Walter Heiligenberg,
who provided the initial inspiration for this project. This
work was supported by NIMH R37 MH26149-18, NINCDS
RO1 NS22244-08, and NSF BNS-9106705 to Dr. Walter
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electric knifefish, Eigenmannia: A quantitative analysis. Brain Res.
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neuroendocrine, interactions, virescens, electric, efferent, system, afferent, diencephalic, weakly, prepacemaker, electromotor, eigenmannia, axis, connection, fish, nucleus
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