An HRP Study of Afferent Connections of the Supracommissural Ventral Telencephalon and the Medial Preoptic Area in Him6 Salmon (Landlocked Red Salmon,
Oncorhynchus nerka)
TAKASHI SHIGA 1,* , YOSHITAKA OKA l, MASAHIKO SATOU l , NAOTO OKUMOTO z and KAZUO UEDA 1
I Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113 and ZNational Research Institute of Aquaculture, Nikko Branch, Nikko, Tochigi 321-16 (Japan)
The supracommissural ventral telencephalon and the medial preoptic area have been shown to play important roles in the sexual be- havior of him6 salmon (landlocked red salmon, Oncorhynchus nerka) 62~63. In the present study, the sites of neurons projecting to these regions were examined by means of the retrograde horseradish peroxidase (HRP) tracing method. The morphology of neurons in these sites of origin was also studied by means of the Golgi method. The nucleus preopticus periventricularis and the rostral part of nu- cleus preopticus (NPP-rNPO) received bilateral projections from the middle parts of the area ventralis telencephali pars ventratis (Vv) and the area ventralis telencephali pars dorsalis (Vd), NPP and lateral part of the preoptic area (LPOA), ipsilateral projections from the caudal part of Vv, nucleus anterioris periventricularis (NAPv), nucleus ventromedialis thalami (NVM) at the level of the pos- terior commissure, nucleus lateralis tuberis pars medialis (NLTm), nucleus anterior tuberis (NAT), nucleus saccus vasculosus (NSV), nucleus recessus posterioris (NRP) and midbrain tegmentum (TG), and a projection from the nucleus posterior tuberis (NPT), which is situated on the midline of the brain. The area ventralis telencephali pars supracommissuralis and neighboring caudal ventral telen- cephalon (Vs-cV) received ipsilateral projections from almost all parts of the Vv, the middle and caudal parts of Vd. almost all parts of the NNP, the NPO at the level between the habenula and the posterior commissure, and the rostral part of the nucleus dorsomedialis thalami (NDM). The Vs-cV also received a projection from NPT. These findings seem to give anatomical bases for understanding the neural mechanisms involved in sexual behavior as well as neuroendocrine functions.
INTRODUCTION
In teleosts it has been suggested, by behavioral ex-
periments using brain ablation, brain lesion and elec-
trical stimulation techniques, that the telencephalic
hemisphere29,34-36.54.65,66 and/or preoptic area
(POA) 12,15,34 play(s) an important role in male sexual
behavior (for reviews, see refs. 8 and 11). In the him0
salmon, we ourselves have shown, by brain lesion
and electrical stimulation experiments, that the area
ventralis telencephali pars supracommissuralis and
the neighboring caudal ventral telencephalon (Vs-
cV), as well as the nucleus preopticus periventricu-
laris (NPP) of the POA, are involved in the control of
male sexual behavior 62,63.
The teleost P O A is also thought to have a variety
of functions in maintaining basic living activities,
such as: (1) regulation of body temperature because
of the presence of thermosensitive neurons41:2; (2)
osmoregulation because of the presence of vasotocin
or isotocin-containing neuronsZt.61; and (3) regula-
tion of the hypophysiogonadal system because of the
presence of the luteinizing hormone-releasing hor-
mone (LH-RH) immunoreact ive neurons 38, sex ste-
roid hormone concentrat ing neurons 9,t°,32,33, and the
gonadotropin release-inhibitory factor (GRIF) ss.
To elucidate the brain mechanisms of sexual be-
havior, and also the autonomic and endocrine func-
tions, it is indispensable to know the neural connec-
tions of these brain regions, However, there have
* Present address: Department of Anatomy, Yamagata University, School of Medicine, Zao, Yamagata 990-23, Japan. Correspondence: K. Ueda, Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan.
only been a few studies of these connections1,2,45,46, 68,
all of which have used conventional histological methods. A reexamination using recent experimen- tal methods is necessary to determine the exact sites of the origin and termination of the fiber systems sug- gested by these classical studies. We thus far exam- ined the efferents from the Vs of him6 salmon using the anterograde degeneration methods and showed that the Vs has large intratelencephalic and extrate- lencephalic proj ections 69.
In the present experiments, we investigated the sites of origin of the afferent projections to the Vs-cV and POA of him6 salmon using the retrograde horse- radish peroxidase (HRP) tracing method. The mor- phology of neurons in these sites of origin was also studied by means of the Golgi method.
MATERIALS AND METHODS
Fish Sixty-eight male him6 salmon (landlocked red
salmon, Oncorhynchus nerka) were used. Fifty-sev- en of them (27.5-34.5 cm in body length) were cap- tured at the mouth of a river flowing into Lake Chu- zenji (Nikko City, Japan) in September and October during the homeward migration about 3 years after hatching. These fish were used for the HRP and gen- eral histological preparations. The remaining 11 fish (22.0-28.0 cm in body length) were obtained from May to July in a pond of the National Research Insti- tute of Aquaculture where they had been cultured for about 3 years. They were used for the HRP study.
Horseradish peroxidase injection Fifty-six fish were used for the HRP study. They
were anesthetized by immersion in a 0.03% tricaine methanesulfonate (MS222) solution and positioned in a specially designed stereotaxic apparatus. Sur- gery was performed during gill perfusion with aer- ated water or 0.01% MS222 solution. The dorsal cra- nium was removed with a razor knife to expose the brain. A glass micropipette (tip diameter, 10-30/~m) filled with 10-40% solution of the HRP (TOYOBO, Grade IC) was driven through the dorsal part of the telencephalon into the Vs or the NPP by a microma- nipulator. A current generator was connected to a sil- ver wire inserted in the micropipette and to another wire attached to the body surface. For iontophoretic
163
HRP delivery, a positive DC of 1.0-2.0/~A, 1 s
on-1 s off, was then applied to the micropipette dur-
ing 5-10 min total on time. Following the iontopho- resis, a negative DC of 1.0-2.0 aA, 1 s on-1 s off, was applied during 2-3 min total on time to prevent the HRP leakage during a retraction of the micropi- pette.
Horseradish peroxidase histochemistry Animals were postoperatively maintained at 9 °C.
Two hours to 10 days after the HRP injection, they were perfused through the conus arteriosus with 0.7% NaC1 solution containing 5 IU/ml heparin, fol- lowed by the primary fixative containing 1% para- formaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and then the secondary fixative containing 2.5% glutaraldehyde and 10% su- crose in 0.1 M phosphate buffer (pH 7.4). Immedi- ately afterwards the brains were dissected out and immersed in 0.1 M phosphate buffer containing 30% sucrose for 12-24 h. Serial frontal sections were cut at 40-60 am on a freezing microtome and mounted on gelatinized slides. The sections were reacted with tetramethylbenzidine after Mesulum 37 and counter- stained with neutral red.
Nissl and Bodian preparations After transcardiac perfusion with 10% formalin sa-
line, the brains of 4 fish were dissected out, em- bedded in paraffin (Paraplast, Sherwood) and sec- tioned frontally at 15/~m. Three of them were stained
Fig.2
FieF4 A B C D E F G H I J K L M N
A B C O E FG H I
o
Fig. 1. Lateral view of the brain of him6 salmon showing the levels of the frontal sections in Figs. 2 and 4.
164
with 0.1% cresyl violet and the remaining one was stained according to the Bodian-Otsuka method53.
Rapid Golgi preparation The brains of 8 other fish, which had been trans-
cardially perfused with 10% formalin saline, were re-
moved from the skull and cut into 3-5 mm thick fron-
tal slices. They were immersed in a rapid Golgi solu-
tion (a mixture containing 3% potassium dichromate
and 0.2% osmium tetroxide) for 2-6 days and then in
a 0.75% silver nitrate solution for 24 h. After impreg-
nation, the slices were dehydrated and embedded in celloidin and sectioned frontally at 75-150
/~m in thickness.
The terminology of the brain areas and nuclei was
mainly after Northcutt and Davis -s0 for telenceph-
alon, and Billard and Peter5 and Nieuwenhuys and
Pouwels 4s for diencephalon and mesencephalon.
RESULTS
Sites of origin of afferent projections to the nucleus preopticus periventricularis and the rostral part of the nucleus preopticus (NPP-rNPO )
We examined various survival periods between 2 h
and 9 days. The distribution of retrogradely labeled
cells was basically identical among the fish which
were allowed to survive more than 6 days after the
HRP injection. In the fish which were killed within 5
days, however, some regions lacked labeled cells (for
example, the midbrain tegmentum), perhaps be- cause of an insufficient amount of the retrogradely
transported HRP. The meninges covering the ventral
surface of the telencephalon and diencephalon
showed HRP-positive reaction, perhaps because of
endogenous peroxidase.
01~
Od~DIvdc~
AC
oa ~\D~d ~ :OA N[~
NOM G
.
Fig. 2. A-N: Nissl-stained sections (left) and line drawings of frontal sections (right) illustrating the distribution of labeled cells (dots) following the HRP injection into the NPP-rNPO (shaded area in E and F). Each dot represents from two to five retrogradely labeled cells. The levels of the sections are shown in Fig. 1. Fish No. 40.
.~" : :.~.;, ~..~-
hiLT m
165
I 1 r a m I
Injection site The POA of him6 salmon consisted of a medial
part (MPOA), where the cells were densely packed, and a lateral part (LPOA), where the cells were sparsely distributed (Fig. 2E-H) . The MPOA was further divided into the NPP, the NPO, and the nu- cleus anterioris periventricularis (NAPv).
In 11 fish, the injected HRP was restricted within the MPOA unilaterally, while in the remaining 15 fish the HRP was further diffused to the LPOA and the neighboring telencephalic hemisphere on the same side. The following results (from Fish No. 40; survival period, 7 days) are typical of the injections in which the HRP was restricted to the region ranging from the ventral part of the NPP to the rostral part of the NPO (shaded areas in Fig. 2E, F).
Distribution of labeled cells The distribution of retrogradely labeled cells is il-
lustrated in Fig. 2. Each dot in the figure indicates the location of retrogradely labeled cells, representing from two to five cells.
Telencephalon. Retrogradely labeled cells were observed bilaterally in the ventral telencephalic nu- clei, Vv and Vd (Fig. 2A-C). There was a tendency for more cells to be labeled ipsilaterally to the injec- tion site than contralaterally. A typical example of the distribution pattern of the labeled cells in the Vv and Vd is illustrated in a histogram (Fig. 3; Fish No. 40). The distribution patterns were essentially iden- tical in all cases, although the total number of labeled cells varied from fish to fish. The Vv contained more labeled cells than the Vd. In the case of Fig. 3, the ip- silateral and contralateral Vv contained 50 and 4 cells,
166
respectively, while the ipsilateral and contralateral Vd contained 15 and 1 cells respectively. Along the
rostrocaudal axis, the following tendency was recog- nized: the labeled Vv cells were distributed in the middle (Figs. 2A, B and 3) and caudal parts (Figs. 2C
and 3) of the ipsilateral side, while the labeled Vd cells were relatively restricted to the middle part of
the ipsilateral side (Figs. 2B and 3). On the contralat- eral side, labeled Vv and Vd cells were seen only in
the middle parts (Fig. 3). The somata of the retrogradely labeled cells were
small and spherical (about 10/~m in diameter). Most
of them were unipolar, and a few were bipolar, but
1700
1000
500
most
r
rostra] Vv
Vv . . . . Vd
0! 2 i i 2 3 4 5 D AC
contra]atera] ipsilateral
Fig. 3. A histogram of the distribution of labeled cells in the Vv (solid line) and Vd (broken line) following the HRP injection into.the NPP-rNPO. The ordinate shows the rostrocaudal dis- tance from the anterior commissure to the most rostral Vv. The abscissa shows the number of labeled cells in the telencephalic hemisphere ipsilateral (right) and contralateral (left) to the in- jection site. Fish No. 40.
none were multipolar (Fig. 5A). For example, in Fish No. 38 (survival, 7 days), there were 25 unipolar and 5 bipolar cells. Their dendrites terminated within the
Vv and Vd without showing any preferred direction. In Golgi-impregnated preparations, most neurons
in the Vv (Fig. 7, neurons 2 and 3) and Vd (Fig. 7, neuron 1) were small in diameter (10-15 Bm). Some
neurons were unipolar and extended a dendrite with no or few spines on the surface (Fig. 7, neurons 1 and 3). The dendrites were often oriented laterally with-
out showing any preferred direction. The HRP-label- ed cells seem to correspond to these neurons. In Gol- gi preparations, other muttipolar neurons were ob- served (Fig. 7, neuron 2) which were not retrograde- ly labeled by the HRP.
Diencephalon and mesencephalon. (1) POA: label- ed cells were observed bilaterally in the POA, al- though more cells were seen ipsilaterally than contra-
laterally. In the ipsilateral MPOA, the NPP (Fig. 2E, F) and NAPv (Fig. 2H) contained labeled cells. The labeled cells in the NAPv were restricted rostrally to the habenula. The ipsilateral LPOA also contained labeled cells (Figs. 2 E - H and 5B). Contralaterally to the injection site, the NPP and LPOA contained la- beled cells. Most of the labeled cells in these regions
were small and unipolar. (2) Nucleus lateralis tuberis (NLT): the NLT was divided into two parts, the ven-
trolateral part, consisting of large neurons (pars lat- eralis, NLTI) and the periventricular part, consisting of small neurons (pars medialis, NLTm). The NLTm of himd salmon seems to correspond to the nucleus
lateral tuberis pars anterioris, pars inferioris, and pars posterioris of goldfish (PeterS6). Labeled cells were observed in the ipsilateral NLTm both at the level of the posterior commissure (Figs. 2I and 5C) and at the level of the nucleus posterior tuberis (NPT) (Figs. 2K and 5D). These cells had small spherical somata and a dendrite extending dorsolat- erally. In the Golgi preparations, a similar type of cell was seen in the NLTm (Fig. 7, neuron 8). These cells had small and spherical somata (8-15/~m in di- ameter) and a sparsely spiny dendrite which branched and ran dorsolaterally. Cerebrospinal fluid contacting neurons were impregnated in Golgi prep- arations (Fig. 7, neuron 9). This type of neuron was not labeled by the HRP. (3) Nucleus posterior tube-
ris (NPT): labeled cells were seen in the NPT; they extended their dendrites laterally and dorsally (Figs.
167
Vd Vv
Vd-
V v -
t DI~Dp " .~~d~-DId
NPP t
NPO
AC ~ I ~ L G NAPv
l m m I I 1ram I I
TL
FR
Fig. 4. A-I: line drawings of frontal sections illustrating the distribution of labeled cells (dots) following the HRP injection to the Vs- cV (shaded areas in D and E). The levels of the sections are shown in Fig. 1. Fish No. V35.
2K, 5E, F). In the Golgi preparations, cells which re- sembled the HRP-labeled cells were impregnated (Fig. 7, neurons 10 and 11). The medium-sized soma- ta were spherical (about 15 pm in diameter) or oval (about 10 x 20 pm), and the spiny dendrites ran either dorsally or laterally. (4) Other diencephalic nuclei: large labeled cells were seen ipsilaterally in the nucleus ventromedialis thalami (NVM) at the level of the posterior commissure (Fig. 21). Small cells were labeled in the ipsilateral nucleus anterior tuberis (NAT) (Fig. 2J), nucleus saccus vasculosus (NSV) and nucleus recessus posterioris (NRP) (Fig. 2L). (5) Midbrain tegmentum (TG): large crescent- or spindle-shaped cells were observed in the ipsilater- al TG medial to the tractus mesencaphalocerebellaris anterior (TMC) (Figs. 2M, 5G, H), and medial to the lemniscus lateralis (LL) (Fig. 2N). In Golgi prepara- tions, similar types of large (15 x 50/~m) crescent- or spindle-shaped cells were seen medial to the TMC, extending very thick aspiny dendrites (Fig. 7, neuron 12).
Sites of origin of afferent projections to the area ven- tralis telencephali pars supracommissuralis and the neighboring caudal ventral telencephalon (Vs-cV)
Various survival periods (5 h-10 days) were exam- ined in 30 animals. With survival periods shorter than 4 days, some regions (for example, NPT) contained no labeled cells probably because of insufficient amount of retrogradely transported HRP. However, the distributions of the labeled cells were essentially identical among the fish which were allowed to sur- vive over 5 days.
Injection site In 9 fish out of 30, the HRP injection site was re-
stricted within the border of the ipsilateral Vs-cV. In the remaining 21 fish, the HRP further invaded neighboring dorsomedial parts of the telencephalic hemisphere. In the latter fish, retrogradely labeled cells were found in the ipsilateral nucleus preglome- rulosus (NPG) in addition to the regions mentioned
168
below. Fig. 4 illustrates one of the typical results ob-
tained from a fish (Fish No. V35; survival period, 6
days) in which the HRP was restricted to the ipsilat-
eral Vs and rostrally neighboring areas, including the
dorsal half of the caudal Vv and the ventral part of
the caudal Vd (shaded areas in Fig. 4D and E).
Distribution of labeled cells Telencephalon. Labeled cells were found in the ip-
silateral Vv and Vd (Figs. 4 A - D and 6A). In the Vv,
labeled cells were observed from the rostral end of
the Vv to the level of the injection site, while in the
Vd, labeled cells were seen from the level in the ros-
Fig. 5. A-H: photographs of the labeled cells following the HRP injection into the NPP-rNPO, counterstained with neutral red. A: la- beled cells (arrowheads) in the Vv at the level corresponding to Fig. 2B. Scale bar, 50~m. B: labeled cell in the LPOA at the level cor- responding to Fig. 2G. Scale bar, 50/~m. C: labeled celt (arrowhead) in the NLTm at the level corresponding to Fig. 2I. Scale bar, 30 /~m, D: labeled cell in the NLTm at the level corresponding to Fig. 2K. Scale bar, 50pro. E: labeled cell (arrowhead) in the NPT at the level corresponding to Fig. 2K. Scale bar, 100pm. F: higher magnification of the labeled cell in E. Scale bar, 50pm. G: labeled cell (ar- rowhead) in the TG at the level corresponding to Fig. 2M. Scale bar, 100pro. H: higher magnification of the labeled cells in G. Scale bar, 50/~m.
169
tral Vd (about 200 gm caudal to the rostral end of the labeled Vv) to the level of the injection site. The la- beled cells had small somata (about 10 gm in diame- ter) and were mostly unipolar. Most dendrites termi- nated within the Vv and Vd; the longest of them could be traced to about 270#m. The dendrites ran in various directions.
Diencephalon. (1) POA: Labeled cells were ob- served in almost all parts of the ipsilateral NPP (Figs. 4F and 6B). They could be classified into two types. The first type extended one or two dendrites laterally into the LPOA. The second cell type was bipolar, and its dendrites coursed dorsoventrally, parallel to the interhemispheric ventricle (Fig. 6B). In Golgi preparations, at least 5 types of neurons could be identified in the NPP. The first type (multipolar neu- ron) was distributed mainly in the most ventral part of the NPP and had a small soma (about 10 gm in di- ameter) and aspiny dendrites running in various di- rections (Fig. 7, neuron 4). The second type (later-
ally orienting neuron) had a spherical soma of small or intermediate size (10-15 ~m in diameter) and den- drites with few spines extending far into the LPOA (Fig. 7, neuron 5). The third type (bipolar neuron) had an oval or spindle-shaped soma of intermediate size (10 × 20/~m) and dendrites with few spines run- ning dorsoventrally along the interhemispheric ven- tricle (Fig. 7, neuron 6). The fourth type (cerebrospi- nal fluid-contacting neuron) had an oval or spindle- shaped soma of intermediate size (10 x 15-20/~m) and one of the dendrites extended medially to con- tact the ventricular surface. The fifth type (commis- sural neuron) had an oval or spindle-shaped soma of intermediate or large size (10-15 x 20-30/~m), and one of the dendrites and an axon invaded the contra- lateral POA. The first and second types of HRP-la- beled cells seem to correspond to the second and third types of Golgi-impregnated cells respectively. The ipsilateral NPO contained large labeled cells (Figs. 4H and 6C). Although the NPO is a rostrocau-
Fig. 5 continued
170
dally long nucleus ranging from the level of the optic
chiasm to the level immediate ly rostral to the posteri-
or commissure, the labeled cells were all found in the
region caudal to the habenula. (2) Other diencephal-
ic nuclei: small, spherical labeled cells were observed
ipsilaterally in the most rostral part of the nucleus
dorsomedial is thalami (NDM) (Fig. 4G). In Golgi
prepara t ions , similar small spherical neurons (about
Fig. 6. Photomicrographs of the labeled cells following the HRP injection into the Vs-cV, counterstained with neutral red. Scale bar 50/zm. A: labeled cells (arrowheads) in the Vv and Vd at the level corresponding to Fig. 4B. B: labeled cell in the NPP at the level cor. responding to Fig. 4F. C: labeled cell in the NPO at the level corresponding to Fig. 4H. D: labeled cell in the NPT at the level corre- sponding to Fig. 4I.
171
11
v ~ j oop j
Fig. 7. Camera lucida drawings of the Golgi-impregnated cells in the Vd (1), Vv (2 and 3), NPP (4, 5 and 6), NDM (7), NLTm (8 and 9), NPT (10 and 11) and TG (12).
8/~m in diameter) were observed with some aspiny dendrites coursing in all directions (Fig. 7, neuron 7).
Labeled cells of the intermediate size were distrib- uted in the NPT (Figs. 41 and 6D). The labeled cells were similar in shape to those observed following the HRP injection into the NPP-rNPO.
The results of the present study are summarized in the schematic drawings in Fig. 8.
DISCUSSION
The present HRP study showed that the NPP- rNPO received bilateral projections from the NPP, LPOA, and middle parts of Vv and Vd, ipsilateral projections from the caudal part of Vv, NAPv, NVM, NLTm, NAT, NRP, NSV and TG, and a pro- jection from the NPT, which is situated on the mid- line of the hypothalamus. On the other hand, the Vs- cV received ipsilateral projections from almost all parts of Vv and NPP, the middle and caudal parts of Vd, NPO at the level between the habenula and the
posterior commissure, and the rostral part of NDM, and a projection from the NPT (Fig. 8).
Technical remarks
To interpret the present results properly, it should be checked whether or not some cells were labeled by incorporating the HRP from the injured fibers pass- ing through the injection site, not from the axon ter- minals. Especially, possible HRP uptake from the lateral and medial forebrain bundles (LFB and MFB respectively), through which both telencephalofugal and telencephalopetal fibers run, should be consider- ed here. At the level of the POA, both forebrain bun- dles were observed to run in the LPOA lateral to the NPP and the NPO in Bodian preparations of the him6 salmon brain. Therefore, the HRP uptake from the forebrain bundles seems improbable at this level. At the level of the Vs-cV the LFB was seen to run lateral to the Vs-cV, while the MFB was too sparsely distrib- uted to be exactly located in Bodian preparations. In the goldfish, Oka and Ueda52 examined the courses
172
A. N P ~
( NAPv "~NPT " ~
B. V s - cV
Fig. 8. Schematic illustration of afferent connections to the NPP-rNPO (A) and to the Vs-cV (B).
of the telencephalopetal fibers through the forebrain bundles by the anterograde degeneration method; they observed no degenerating fibers in the ventral telencephalon including the Vs-cV. In the coastal rockfish, Murakami et al. 40 examined the efferents from the dorsal parts of the telencephalon and ob- served that the forebrain bundles do not run through the Vs; the MFB runs just ventral to the Vs, while the LFB runs far lateral to the Vs. Therefore, it seems that there are no LFB fibers and probably few, if any, MFB fibers running through the Vs-cV of the him6 salmon. Furthermore, we used capillaries with small tip diameters (10-30/~m) in order to reduce the pos- sibility of injuring the passing fibers at penetration. Thus, the possible uptake of HRP from passing fibers seems to be avoided, and most of the HRP-labeled cells in the present study can be regarded as afferent neurons terminating within the HRP-injected re- gions.
Afferent connections of the ventral telencephalon and the preoptic area
Concerning the afferent system of the ventral tel- encephalon and MPOA, there have been only a few classical anatomical studies (e.g. Ari6ns Kappers et al. l). Although the neural connections of the fish tel- encephalon have recently been studied using HRP, anterograde degeneration and autoradiographic methods, all of the studies have focused on the dorsal parts of the telencephalon, not on tile ventral parts of the telencephalon 17:9.2v.4(L49 (for reviews, see refs. 47 and 50).
According to the conventional histological studies concerning the fiber connections between the telen- cephalon and POA, the ventral parts of the telen- cephalon (area ventralis telencephali) send fibers to the POA 2,45.46,68. This agrees well with the present
finding that the Vv and Vd project bilaterally to the NPP-rNPO.
It has been reported that the electrical stimulation of the dorsal parts of the telencephalon (area dorsalis telencephali) activates the neurons of the NPP in sun- fish 22. We could not find any direct projection from the area dorsalis telencephali to the NPP-rNPO. It is probable that the NPP-rNPO receives inputs from the area dorsalis telencephali via neurons in area ventralis telencephali, since projections have been demonstrated from area dorsalis telencephali to area ventralis telencephali 4° and from area ventralis telen- cephali to the NPP-rNPO (present study). However, there is a possibility that the NPP-rNPO neurons re- ceive direct inputs from the area dorsalis telencephali via their dendrites in the LPOA (see below).
Sensory inputs to the ventral telencephalon and the
preoptic area It has also been reported that the area ventralis tel-
encephali and POA receive some sensory inputs, such as visual or olfactory.
The retina sends a direct projection to the MPOA in goldfish 67,71 and him6 salmon (unpublished obser- vation). The direct projection from the retina to the LPOA, where the MPOA extends their dendrites, has also been reported in many teleosts (for a review, see ref. 74). Therefore, it seems likely that the MPOA receives direct visual input.
The afferent connections from the olfactory bulb to the area ventralis telencephali have been shown in
several teleosts by anatomicaP, 7,18,25,26,40,51.64 and
electrophysiological methods 20. Although we could not find any direct projection from the olfactory bulb to the Vs-cV, we found retrogradely labeled cells in the olfactory bulb when the injected HRP diffused as far as the rostral part of the ventral telencephalon. Therefore, it is probable that the Vs-cV receives ol- factory input via neurons in the rostral part of the ventral telencephalon. Moreover, the present study has shown that the Vs-cV receives afferents from the NPT. Since the projection from the olfactory bulb to the NPT has been reported in several teleosts3, 7, 18,20,40, it is also probable that the Vs-cV receives ol-
factory input via the NPT. The POA has also been shown, by anatomical3, 40,
electrophysiological 4,2°,22,23,3°, and neuroendocrino- logical methods28,57.59. 60, to receive afferents from the olfactory bulb through the olfactory tract. In the present study, we could not recognize any direct pro- jections from the olfactory bulb to the NPP-rNPO. However, it is probable that the NPP-rNPO receives olfactory input via neurons in the area ventralis tel- encephali or NPT, since the projections from the ol- factory bulb to the area ventralis telencephali3, 7, 18.20.25.26.40.51.64, and to the NPT3,7As,20, 40, and the pro-
jection from the area ventralis telencephali and NPT to the NPP-rNPO (the present study) have been demonstrated. In addition, the olfactory bulb of the carp sends fibers to the dorsal part of LPOA, the fi- ber-rich zone dorsolateral to the NPP 20. The present Golgi and HRP study has revealed that some neurons in the NPP also extend dendrites into the LPOA. Therefore, it seems also possible that neurons of the NPP receive direct olfactory projection via their den- drites in the LPOA.
In connection with the olfactory system, it may be worthwhile to discuss the nervus terminalis system. In the goldfish where the olfactory bulb is separated from the telencephalic hemisphere by the long olfac- tory tract, the ganglion cells of the nervus terminalis, which are either interspersed between the olfactory nerve fibers or rostrally along the ventromedial as- pect of the olfactory bulb, project to the vicinity of the anterior commissure and POA16, 70. On the other hand, in fishes which have sessile olfactory bulbs, such as the cichlid, poecilid, and centrarchid, the ax- ons of the neurons in the nucleus olfactoretinalis (NOR), which is located at the transitional area be-
173
tween the area ventralis telencephali and the olfacto-
ry bulb, run through the area near the anterior com- missure 39. Both the nervus terminalis and NOR con- tain LH-RH-immunoreactive neurons and neurons projecting to the retinal6,38,39,70,73; thus, they might
be homologous with each other 39. In order to identify the NOR in him6 salmon which has sessile olfactory bulbs, we applied HRP to the proximal end of the cut optic nerve. Several retrogradely labeled cells were sparsely distributed in the contralateral region lateral to the Vv, though some were in the lateral part of the Vv, while anterogradely labeled axons ran in the Vs and LPOA (unpublished observation). In the present study, in which the HRP was injected into the Vs-cV or NPP-rNPO, retrogradely labeled cells were ob- served in the lateral part of the Vv, but not in the re- gion lateral to the Vv. Therefore, there is a possibili- ty that a part of the NOR neurons in the lateral part of the Vv project to the Vs-cV and NPP-rNPO, al- though further experiments are needed to clarify this point. It has been suggested that the ganglion cells of the nervus terminalis in the goldfish may be con- cerned with sperm release13,16. As has been men-
tioned above, it is~possible that both the Vs-cV and NPP-rNPO receive input from the medial part of the NOR, which might be homologous with the nervus terminalis.
Relevance to the sexual behavior
Recent behavioral experiments, such as brain ab- lation, brain lesion and electrical stimulation experi- ments, have shown that the telencephalic hemi- sphere and MPOA play some facilitatory roles in the male sexual behavior of several teleosts12A 5, 29,34-36,54,65,66. It has also been shown in him6 salmon
that the Vs-cV and NPP play important roles in male sexual behavior62, 63.
It has been reported that some kinds of visual in- formation from the female facilitate the male sexual behavior of the rainbow trout 44. Because the NPP may receive visual input, as has been mentioned above, there is a possibility that the NPP is involved in the control of the male sexual behavior by process- ing visual information.
It has also been reported that pheromones re- leased from the ovulated female act on the male as an olfactory stimuli for the elicitation of male sexual be- havior in rainbow trout24, 43 and in goldfish55, 72. Since
174
the Vs-cV and NPP-rNPO of him6 salmon may re- ceive olfactory inputs, as has been mentioned above,
it is possible that these regions are involved in the
control of male sexual behavior by processing olfac-
tory information.
In several teleosts, castration reduces sexual be-
havior while androgen restores castration-reduced
sexual behaviors (for reviews, see refs. 14 and 31). In
this respect, it is interesting that the Vs-cV and NPP-
rNPO, as well as NPP, NPO, L P O A , NLTm, NVM
and NSV, which were shown, in the present study, to
project to the Vs-cV or NPP-rNPO, accumulate sex steroid hormones in several teleosts 9A0,32,33. Sponta-
neous activities of the neurons in the M P O A and me-
dial hypothalamus of the female rat are affected by
estrogen 6. Accordingly, it is possible that neurons in
the Vv-cV and NPP-rNPO, as well as the regions pro-
jecting to them, are involved in the control of male
sexual behavior under the influence of sex steroid
hormones, possibly by controlling the level of sexual
motivation.
The present study has revealed that there are com-
mon sources of afferents to the Vs-cV and NPP-
rNPO, as well as different afferents. Both regions re-
ceived afferents from the Vv and Vd ipsilaterally,
and from NPT, while the Vs-cV, but not the NPP-
rNPO, received ipsilateral projections from the ros- tral Vv, NDM and NPO, and the NPP-rNPO, but not
the Vs-cV, received projections from the contralat-
eral Vv and Vd, NAPv, LPOA, NVM, NAT, NLTm,
NRP, NSV and TG. Moreover, fiber connections be-
tween the NPP-rNPO and Vs-cV were not recip- rocal, but unidirectional (from NPP to Vs-cV).
Some functional differences in the control of male
sexual behavior have been suggested between the Vs-cV and NPP of him6 salmon62. 63. A weak electri-
cal stimulation of the NPP of the male him6 salmon
elicits courtship behavior toward the female (quiver-
ing), while stimulation with a stronger intensity evokes the spawning act and sperm release. On the
other hand, the electrical stimulation of the Vs-cV evokes spawning act and sperm release, but not the courtship behavior (quivering).
It is possible that these differences in the effects of electrical stimulation are, at least in part, attributed
to the differences in the afferent systems mentioned
above, in addition to the differences in the efferent systems.
ACKNOWLEDGEMENTS
The authors wish to thank Mr. T. Shimada, Na-
tional Research Institute of Aquaculture, Nikko
Branch, for taking care of the fish, and the staff of the
Lake Chuzenji Fishery Association for providing the fish and the facilities for the experiment.
ABBREVIATIONS
AC Dc Dd Dld Dlv Dm Dp FLM FR HOC Hyp LI LL LPOA LV MO MPOA NAPv NAT NDL NDLI NDM
anterior commissure area dorsalis telencephali pars centralis area dorsalis telencephali pars dorsalis area dorsalis telencephali pars lateralis dorsalis area dorsalis telencephali pars lateralis ventralis area dorsalis telencephali pars medialis area dorsalis telencephali pars posterioris fasciculus longitudinalis medialis fasciculus retroflexus horizontal commissure hypophysis lobus inferioris lemniscus lateralis lateral preoptic area nucleus lateralis valvulae medulla oblongata medial preoptic are nucleus anterioris periventricularis nucleus anterior tuberis nucleus dorsolateralis thalami nucleus diffusus lobi inferioris nucleus dorsomedialis thalami
NDTL nucleus diffusus tori lateralis NE nucleus entopeduncularis NG nucleus glomerulosus NGL nucleus geniculatus laterale NLTI nucleus lateralis tuberis pars lateralis NLTm nucleus lateralis tuberis pars medialis NNO nucleus nervi oculomotorii NPG nucleus preglomerulosus NPO nucleus preopticus NPP nucleus preopticus periventricularis NPPv nucleus posterioris periventricularis NPT nucleus posterior tuberis NR nucleus rotundus NRL nucleus recessus tateralis NRP nucleus recessus posterioris NRu nucleus ruber NSV nucleus saccus vasculosus NVM nucleus ventromedialis thalami OB olfactory bulb OC optic chiasma ON optic nerve OT optic tract PC posterior commissure POA preoptic area
area ventralis telencephali pars commissuralis area ventralis telencephali pars dorsalis area ventralis telencephali pars iateralis area ventralis teleneephali pars postcommissuralis area ventralis telencephali pars supracommissuralis area ventralis telencephali pars ventralis
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