Cell Tissue Res (1990) 259:543-550

and T' sue

© Springer-Verlag 1990

The caudal spinal cord of coho salmon (Oncorhynchus kisutch): Immunocytochemical evidence of a "caudal serotoninergic system" Carlos R. Yulis, Maria E. Garcia, and Esteban M. Rodriguez Instituto de Histologia y Patologia, Universidad Austral de Chile, Valdivia, Chile

Summary. The caudal spinal cord of the coho salmon was investigated by means of immunocytochemistry using anti- sera against serotonin, urotensin I, urotensin II, somatosta- tin and a urea-extract of bovine Reissner's fiber (AFRU). Populations of serotonin-immunoreactive (IR) neurons were found rostral and dorsal to the urophysis in close spatial association with caudal secretory neurons. Thick, smooth serotonin-IR processes extended toward the exter- nal surface of the spinal cord where they displayed conspic- uous terminal dilatations. Thin, beaded serotonin-IR fibers appeared to innervate populations of caudal secretory and

somatostatin-IR cerebrospinal fluid-contacting neurons. Most caudal neurosecretory cells displayed both urotensin I and urotensin II immunoreactivities; only a minority reacted exclusively with either urotensin I or urotensin II antisera. Urotensin II-IR and somatostatin-IR cerebrospi- nal fluid (CSF)-contacting neurons were found as an inte- gral component of the central canal wall in the caudal spinal cord and ilium terminale; their dendritic processes ap- peared to contact Reissner's fiber, which displayed a weak AFRU-immunoreactivity while inside the central canal, but became strongly reactive in the interior of the terminal ven- tricle as it formed the massa caudalis. The distribution of serotoninergic processes points to a regulatory role in the function of caudal secretory and CSF-contacting neurons and to a putative serotonin release into the subarachnoid space and/or meningeal vasculature. It is also suggested that the CSF-contacting neurons of the central canal may participate in a feedback mechanism controlling the secreto- ry activity of the subcommissural organ.

Key words: Serotonin - Urotensins - Somatostatin - Im- munocytochemis t ry- Caudal neurosecretory system - Reissner's fiber (subcommissural organ) - Salmon, Onco- rhynchus kisutch (Teleostei)

The caudal spinal cord of teleosts, elasmobranchs and gan- oids constitutes a region of particular neurobiological inter- est due to the presence of a "caudal neurosecretory sys-

Supported by Grant A/1095-1 from the International Foundation for Science, Sweden, to C.Y. ; Grant 1/63-476 from Volkswagen- Stiftung to E.R. ; and Grant S-85-39 from the Direcci6n de Investi- gaciones, Universidad Austral de Chile

Send offprint requests to: Dr. Carlos R. Yulis, Instituto de Histo- logia y Patologia, Universidad Austral de Chile, Valdivia, Chile

tern". This conspicuous system consists of large-sized secre- tory neurons projecting primarily to a neurohemal area de- scribed as "urophysis" (Bern and Takasugi 1962; Fridberg 1963; Fridberg and Bern 1968). Whereas the physiological role of this well-developed neurosecretory system is still open to discussion, great advances have been made in the chemical identification of the molecular structure of uroten- sins I and II (biologically active peptides synthesized by neurons of the system), and in the morphological analysis of the neuronal components of the caudal neurosecretory system.

Urotensin I (UI), a 41-residue peptide isolated from uro- physes of three teleostean species, shows little variations in their respective sequences (Ichikawa et al. 1982; Lederis et al. 1982; McMaster et al. 1988). It shares a close sequence homology with mammalian corticotropin-releasing factor (CRF, Vale et al. 1981 ; Rivier et al. 1983; Shibahara et al. 1983), fish CRF (Okawara et al. 1988) and sauvagine, a 40-residue peptide isolated from the skin of frogs (Montecu- chi et al. 1980).

Six related forms of urotensin II (UII) have been identi- fied in three teleostean species, i.e., Gillichthys: UII; Cyprin- us: UII~, ,6, 7; and Catostomus: UIIA and B (Pearson et al. 1980; McMaster and Lederis 1983; Ichikawa et al. 1983). All are cyclic dodecapeptides sharing an identical six-resi- due disulfide-bonded ring in positions 6-11.

Immunocytochemical studies on the distribution of cau- dal neurosecretory peptides have revealed colocalization of immunoreactive-UI and -UII in caudal neurosecretory cells of several fish species (Bern et al. 1985; Owada et al. 1985; Yamada et al. 1985; Onstott and Elde 1986a, b; Yulis and Lederis 1986a; Larson et al. 1987). Furthermore, a system of CSF-contacting UII-IR and SOM-IR neurons has been described in some teleost and nonteleost species (Yulis and Lederis 1986a, 1988 a, 1988 b). CSF-contacting UII-IR neu- rons did not display UI-IR and were even present in species in which a caudal neurosecretory system has not been ob- served; thus, these cells can be regarded as belonging to a separate peptidergic system, more common in phylogeny than the caudal neurosecretory system.

Most reports dealing with the distribution of serotonin (5HT)-IR elements in the central nervous system (CNS) of fishes have not considered the situation in the caudal portion of the spinal cord. However, a recent publication describes the presence in this region of a population of 5HT-IR cells that probably constitutes a source of seroton- inergic input to caudal neurosecretory cells in some non- teleost species (Onstott and Elde 1986b).

544

The purpose of the present immunocytochemical study of the caudal spinal cord in O. kisutch, including the filum terminale and ampulla caudalis, was to: (1) investigate the distribution of 5HT-IR perikarya and fibers in this region; (2) search for spatial association between 5HT-IR neuronal structures and elements belonging to the caudal neurosecre- tory and CSF-contacting systems; (3) define the distal ex- tension of caudal neurosecretory, CSF-contacting'and puta- tive 5HT-IR systems; and (4) determine the relationships between the above-mentioned structures and the Reissner's fiber at the caudal end of the central canal.

Materials and methods

Specimens of coho salmon (Oncorhynchus kisutch, n= 14, kindly provided by pisciculture "Mares Australes", Llan- quihue lake, Chile) were used in this study. The fishes were collected from containers made of fishing net kept im- mersed in the lake water. After decapitation, the caudal peduncle was sectioned at the level of the adipose fin; soft tissues were dissected out leaving the vertebral column ex- posed and the caudal fin intact. Time lapse between capture of each fish and beginning of fixation was 5-10 min. Immer- sion fixation was performed in Bouin's fixative (room tem- perature, 2-3 h), followed by washing in PBS (4 ° C, 3 x 20 min) and postfixation in Bouin's fluid without acetic acid (4 ° C, 48 h). In some cases, the spinal cord (including the urophysis and part of the filum terminale) was dissected out after the first fixation period, and then the fixation procedure was continued as described above.

The tissues were embedded in Paraplast. Serial sagittal sections (8 gm) were cut, mounted on gelatin-coated slides and immunostained by the PAP (peroxidase-antiperoxi- dase; Sternberger et al. 1970) method.

Antisera raised against serotonin (5HT; Immuno Nucle- ar Corp, Stillwater, MN, USA), Gillichthys mirabilis UII, Catostomus commersoni UI (both kindly provided by Dr. K. Lederis, University of Calgary, Canada), somatostatin (SOM; kindly provided by Dr. A. Weindl, University of Munich, FRG), and against a urea-extract of bovine Reissner's fiber (AFRU; Rodriguez et al. 1984) were used. The cross-reactivity of the UI antiserum (As) with CRF peptides was prevented by means of solid-phase adsorption using Sepharose 4b beads coupled to ovine CRF (Yulis and Lederis 1986b).

All dilutions of antisera were prepared with a solution containing carrageenan (0.7%) and Triton X-100 (0.1%) in TRIS buffer, pH 7.7.

Reagent concentrations and incubation times were as follows: (1) First As (5HT-As 1 : 1500, UII-As 1:800, UI-As 1 : 500, SOM-AS 1 : 2000, AFRU 1 : 2000) 18 h; (2) Second As (goat anti-rabbit IgG 1:25) 30 min; (3) PAP (peroxidase anti-peroxidase complex, 1:50) 30 min; (4) DAB 0.2% in TRIS buffer containing 0.05% H202, 15 min. TRIS-buffer washings (3 x 5 min) were included between each step.

The primary antisera were applied on adjacent serial sections. For reference, hematoxylin-eosin staining was per- formed with every tenth section.

Results

General aspects

The caudal spinal Cord of coho salmon displayed an elon- gated, slightly prominent urophysis, which was rather diffi-

~ ~ _2-

-AC

Fig. l. Schematic drawing of the caudal spinal cord of the coho salmon showing the position of the urophysis (U), filum terminale (Fir), and ampulla caudalis (AC). The sites at which the subsequent figures were obtained are outlined

cult to distinguish in the gross preparation, even under the dissecting microscope. Caudally, the urophysis was in con- tinuity with a fllum terminale of decreasing diameter, finally surrounding the central canal with an extremely thin tissue layer (Figs. 1, 4). This slender filum terminale extended into the caudal-fin ending, close to its dorsal border, in an elon- gated ampulla caudalis (Figs. 1, 5). The lumen of the central canal, along the spinal cord and filum, was wide enough to harbor Reissner's fiber (Fig. 4h), whereas at the entrance to the ampulla caudalis the lumen expanded, acquiring fea- tures of a terminal ventricle (Fig. 5). The diameter of Reissner's fiber in the central canal of the caudal spinal cord and the filum terminale was rather uniform; in the terminal ventricle, however, the fiber lost its threadlike ap- pearance and gained the configuration of an elongated and conical massa caudalis (Fig. 5 a, c).

Immunocytochemistry

The study of the caudal spinal cord of the coho salmon, by the use of 5HT-As, revealed the presence of two popula- tions of 5HT-IR neurons. One was located rostrally (Fig. 2 a, e), the other dorsally (Fig. 3 a, b) of the urophysis. 5HT-IR bipolar and multipolar neurons displayed round nuclei. These cells were endowed with thick, smooth and thin, beaded processes (Fig. 2e). No colocalization of 5HT with UI or UII was found when adjacent sections were immunostained using the respective antisera. The rostral population of 5HT-IR neurons occupied a plane ventral to the caudal neurosecretory UI-UII - IR neurons observed at the same level (Fig. 2a-c), while in the region dorsal to the urophysis, smaller 5HT-IR neurons occurred inter- mingled with local caudal secretory neurons (Fig. 3a, b). Apart from the spatial relationship of the perikarya, both systems appeared to be linked (i) by scarce 5HT-IR fibers of the thin, beaded type found near to or even surrounding caudal neurosecretory neurons located in the rostral portion of the complex (Fig. 2a, f), and (ii) by a relatively dense net of thin, beaded 5HT-IR fibers surrounding caudal neu- rosecretory cells in the region dorsal to the urophysis. The concentration of fibers forming this net and also the number of 5HT-IR neurons in the region increased toward the level of the caudal border of the gland (Figs. l, 3 a).

Most 5HT-IR thick, smooth processes projected toward the external surface of the caudal spinal cord. Thick, smooth 5HT-IR fibers running in parallel with the external surface of the spinal cord (Fig. 2 e, f) formed a superficial bundle, which became well-defined in the proximity of the urophysis (Fig. 2a). In the region of the "neck" rostral to the urophysis and in the filum terminale, a profuse accu-

545

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Fig. 2 a-c. Serial sagittal sections (a-c) of the pre-urophyseal region immunostained for 5HT, UI and UII, respectively. Note the pres- ence of 5HT-IR perikarya (a small arrows), thin beaded fibers (a thin arrows), and coarse fibers forming a ventral tract (a wide arrow). Extensive fiber dilatations, containing 5HT-IR fine-granu- lar material, aggregated near the external surface of the "neck" region rostral to the urophysis (a, d asterisk). Medium-sized 5HT- IR neurons endowed with several processes (e small arrow) and moderate numbers of thin, beaded 5HT-IR fibers (e, f thin arrows), some encircling caudal neurosecretory neurons (f), in the pre-uro- physeal region. Most thick, smooth fibers are located close to the ventral surface of the spinal cord (a, e, f wide arrows), in parallel with the UI-UII-IR spino-urophyseal tract (b, e wide arrows). At this level of the spinal cord, populations of caudal secretory neu- rons displaying colocalization of UI- and UII immunoreactivities (b, e small arrows), and neurons in which UII immunoreactivity was dominant (e open arrows), were observed. Correspondingly, higher densities of UII-IR thin, beaded fibers (b, e thin arrows) and thick, smooth fibers (b, c wide arrows) were found in this region, a--c x 67, d x 203, e, f× 90

mulation of superficial fiber swellings containing granular 5HT-IR material, was observed (Figs. 2a, d; 4b). In these regions, incoming 5HT-IR fibers of average diameter exhib- ited local widenings measuring up to 15 lam as they ap- proached the external surface of the nervous tissue. Further accumulations of dilated fibers at the external surface of the spinal cord were observed more rostrally, in association with the segmental emergence o f the ventral roots.

The study of adjacent sections of the caudal spinal cord by use of UI -UI I immunocytochemistry revealed a colocali- zation of UI and UII immunoreactivities in most caudal secretory neurons. Neurons displaying exclusively UI I - IR were observed at the rostral aspect o f the caudal neurosecre- tory system (Fig. 2, compare b~) . Neurons exhibiting ei- ther UI I - IR or UI - IR as their main immunoreactivity were

found in the region located dorsal to the urophysis (Fig. 3, compare c-d). There was no indication of differences be- tween the distribution of UI- and UI I immunoreactivities in relation to the perivascular endings in the urophysis (Fig. 3c, d).

Caudally, the population of U I - U I I - I R neurons, recog- nized as belonging to the caudal neurosecretory system, decreased as the filum started to taper (Fig. 4f, g). The most caudal neurons of the system were found at a level between the urophysis and the ampulla caudalis (located in the caudal fin). However, caudal populations o f CSF- contacting UI I - IR neurons (Fig. 4e) and SOM-IR neurons (Fig. 4c) were found in the ventral and dorsal walls o f the central canal, respectively, reaching the level of the rostral border of the ampulla caudalis. Dendrite-like processes of these cells appeared to contact Reissner's fiber inside the central canal. Parasagittal tracts consisting of UII - IR, SOM-IR and 5HT-IR nerve fibers (Fig. 4b, d, f) were also found in the thin wall of the central canal. 5HT-immuno- staining of sagittal sections containing the filum terminale also revealed thin, beaded fibers, distributed among the cells located in the dorsal wall of the central canal. Images sug- gestive of a 5HT-IR innervation of some of these dorsal- wall cells were observed in the filum terminale (Fig. 4a); spatially they corresponded to the sites where CSF-contact- ing SOM-IR neurons formed a palisadelike array (Fig. 4c). No 5HT-IR perikarya were found in the filum terminale or around the ampulla caudalis.

A loose tract of thin, beaded SOM-IR fibers, apparently originating from the above-mentioned CSF-contacting SOM-IR perikarya, could be traced in a parasagittal posi- tion via the structural framework of the caudal spinal cord (Figs. 3 e, 4 d).

At the level of the ampulla caudalis, no structures immu- noreactive with the UI-, UII-, SOM- or 5HT-As used were observed. The material of Reissner's fiber, which was weakly AFRU-immunoreact ive inside the main portion of the central canal (Fig. 4h), displayed an increased immuno-

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Fig. 3 a--e. Sagittal sections through the urophyseal region immuno- stained for 5HT (a, b), UI (c), UII (d), and SOM (e). Note small 5HT-IR, mostly bipolar neurons dorsal to the urophysis (a, b small arrows). These cells are more numerous toward the caudal bound- ary of the urophysis (a small arrows), where numerous 5t!T-IR thin, beaded fibers form a dense network surrounding caudal secre- tory neurons (a thin arrows). Only scattered 5HT-IR processes oc- cur in the vicinity of urophyseal capillaries (b thin arrows). UI- and UII immunostainings of adjacent sections of the urophyseal region (c, d, respectively), show peptidergic neurons displaying both immunoreactivities with equal intensity and other cells in which UI-immunoreactivity is dominant or vice versa (b, c small arrows). A loose tract of SOM-IR thin, beaded fibers extends in a parasagittal plane via the region (d, thin arrows). U urophysis; C capillaries, a x 325, b x 404, c--e x 90

reactivity as it approached the ampul la caudalis. Within the ampulla, Reissner 's fiber lost its threadl ike appearance to form the massa caudalis, which became highly A F R U - immunoreact ive (Fig. 5c). Hematoxyl in-eosin-s ta ined sec- tions revealed an apparent communicat ion of the enlarged ampul la caudalis with a b lood vessel and the presence of

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cells with characteristics of macrophages adhering to the surface of the massa caudalis (Fig. 5 a). The A F R U - I R ma- terial of the massa caudalis was oriented toward the dorso- caudal wall of the ampul la caudalis (Fig. 5c) where the communicat ion with the adjacent b lood vessel was detected. In tangential sections diffuse A F R U - I R mater ial was found to occur between the ependymal cells lining the wall of the ampul la caudalis and the surrounding connective tissue (Fig. 5b, c).

Discussion

The present repor t describes the presence of a popula t ion of 5HT- IR per ikarya in the caudal spinal cord of coho salmon, the distr ibution of which corresponds to that of the bulk of U I - U I I - I R caudal secretory neurons. Mos t of the processes arising from these 5HT-IR neurons appeared to be directed to circumscribed areas in the caudal spinal cord, thus forming a "cauda l serotoninergic sys tem" (Fig. 6). Interestingly, in coho salmon, urophyseal capil lar- ies did not seem to establish a relevant target site for 5HT-

547

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Fig. 4a-h. Sagittal sections of the ilium terminale, at a level be- tween the urophysis and the ampulla caudalis, immunostained for 5HT (a, b), SOM (c, d), UII (e, f), UI (g), and AFRU (h). 5HT-IR thin fibers appear to innervate cells in the dorsal wall of the central canal (a thin arrows), corresponding to the location of SOM-IR CSF-contacting neurons (e short arrows). Scattered UII-IR CSF- contacting perikarya occur in the ventral wall of the central canal (e small arrows)• This is the most caudal level where UII-IR and UI-IR neurons, identified as belonging to the caudal neurosecre-

h

tory system (f, g small arrows), are found. 5HT-IR, SOM- and UII-fibers form parasagittal tracts in the ilium terminale (b, fl, f thick arrows); UI-IR fibers, although present in this region (g thin arrows), do not establish a discrete tract. 5HT-IR terminals close to the external surface of the ilium terminale are shown in a tangential section of the ilium (b thin arrows). Reissner's fiber, located in the narrow central canal of the ilium terminale, displays a weak, mostly peripheral AFRU-immunoreactivity (h). CC central canal; RF Reissner's fiber• a-h x 404

IR secretion. 5HT-IR enlarged, smooth fibers, observed close to the external surface of confined areas of the caudal spinal cord, displayed di latat ions resembling fiber endings. Fur ther evidence of putat ive fiber terminals close to the external surface of fish spinal cord has been provided pre- viously by means of immunocytochemist ry (UI -UI I - IR pro- cesses; Yulis and Lederis 1986a, 1986b, 1988a) and trans-

mission electron microscopy (Vigh et al. 1977), performed at other levels o f the spinal cord of several fish species. These findings, in addi t ion to the evidence presented here, suggest that secretion released from terminals of differing nature arranged at the external surface o f the spinal cord might reach the subarachnoid space and/or the meningeal vasculature and thus be able to act on distant targets.

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Fig. 5a--c. Sagittal sections through the caudal end of filum terminale and ampulla caudalis. Hematoxylin-eosin staining (a) shows the transition of the threadlike Reissner's fiber (RF) into an enlarged massa caudalis (MC) inside the terminal ventricle. Cells resembling macrophages adhere to massa caudalis (a small arrows). The caudal end of the ampulla caudalis appears to be in direct communication with a blood vessel filled with erythrocytes (a, E). A tangential section adjacent to that shown in a, but immunostained with AFRU, shows ampulla caudalis with AFRU-IR material among the ependymal cells and in the adjacent connective tissue (b arrows). AFRU immunostaining in another fish revealed a very strong immunoreactivity of the massa caudalis (c, MC) and of the material scattered in the surrounding connective tissue. a - - c x 147

While most of the 5HT-IR thick, smooth fbers seemed to be projecting toward the external surface of the spinal cord, it appeared that 5HT-IR thin beaded fibers were mainly related to neighboring cells belonging to the caudal secretory or to the CSF-contacting SOM-IR system, in a pattern suggestive of axo-somatic contacts.

By the use of histofluorescence and transmission elec- tron microscopy, it has been shown that monoaminergie, cholinergic and peptidergic terminals establish synaptic contacts with caudal secretory neurons in several fish spe- cies (Audet and Chevalier 1981; O'Brien and Kriebel 1983; Miller and Kriebel 1986). Deafferentation studies revealed that most synapses on caudal secretory neurons originate from descending fiber systems (O'Brien and Kriebel 1983); however, our results in the coho salmon, in addition to the findings of Onstott and Elde (1986b) in some nonteleost fishes, indicate the existence of an intrinsic serotoninergic input to the peptidergic system• The extent of this innerva- tion appears to vary with respect to local populations of caudal secretory neurons in individual animals and also among fish species.

Populations of caudal secretory neurons displaying a variable degree of UI-UII colocalization have been de- scribed also in other fish species (Bern et al. 1985; Owada et al. 1985; Yamada et al. 1985; Onstott and Elde 1986a, b; Yulis and Lederis 1986a; Larson et al. 1987). The recent elucidation of cDNA sequences encoding UI- and UII pre- cursor molecules (Ishida et al. 1986; Ohsako et al. 1986) allowed Ichikawa et al. (1988) to conduct in-situ hybridiza-

tion studies demonstrating a clear-cut colocalization of the respective mRNAs in UI-UII-IR caudal secretory neurons of the carp. These results support previous suggestions that all neurons of the caudal neurosecretory system are capable of synthesizing both UI and UII peptides (Onstott and Elde 1986a, Yulis and Lederis 1986a), and that the visualization of different neuronal populations on the basis of differing intensities of UI- or UII immunostainings (as also reported here) may be the result of independent mechanisms control- ling the turnover of either peptide in the perikarya.

This is the first report showing the presence of UII-IR and SOM-IR CSF-contacting neurons in the filum termin- ale of a teleost fish. Previous reports on the presence of such neurons in the walls of the central canal were focused on a more rostral portion of fish spinal cord (Yulis and Lederis 1986a, 1988a, 1988b). The following findings sug- gest an interrelationship between the caudal serotoninergic neurons, the UII-IR and SOM-IR CSF-contacting neurons, on the one hand, and the subcommissural organ-Reissner's fiber complex, on the other. (1) The impressive distribu- tional pattern of UII-IR and SOM-IR CSF-contacting neu- rons extending from rostral toward the caudalmost levels of the central canal supports the view that these neurons play a significant role with respect to the substrates con- tained in the central canal• At the level of the filum termin- ale, the material of Reissner's fiber appears to be the most prominent intracanalicular constituent. (2) Our results sug- gest that bundles composed of SOM-IR and UII-IR nerve fibers located in the filum terminale originate from CSF-

549

SOM RF CC ~ S=OM

Fig. 6. Diagram summarizing putative neuronal interrelationships in the caudal spinal cord of coho salmon. 5HT-IR thin, beaded fibers innervate caudal secretory neurons and SOM-IR CSF-con- tacting neurons in the filum terminale. These fibers probably origi- nate from: local (caudal) 5HT-IR neurons, serotoninergic neurons located in upper regions of the CNS, or both. 5HT-IR thick,

smooth fibers project to the external surface of the spinal cord and ilium terminale (the last projection is not represented in the diagram). Dendrites of Ull-IR and SOM-IR CSF-contacting neu- rons contact Reissner's fiber (RF) inside the central canal (CC). Thin fibers arising from these CSF-contacting neurons form appar- ently ascending pathways. U urophysis; M C massa caudalis

contacting neurons and subsequently form ascending path- ways conveying information to higher levels of the CNS (Fig. 6). (3) On the other hand, the absence of 5HT-IR perikarya in the ilium terminale suggests that 5HT-IR nerve fibers observed in this region form a part of a descending pathway probably arising from caudal serotoninergic neu- rons (Fig. 6). While thick, smooth fibers of this serotonin- ergic pathway reach the external surface of the ilium termin- ale, thin, beaded axons appear to innervate the above-men- tioned SOM-IR perikarya and therefore might be involved in a response of these CSF-contacting neurons to putative stimuli perceived from inside the central canal (under par- ticipation of Reissner's fiber?). (4) Reissner's fiber is subject to ongoing degradation at its caudal end, followed by con- tinuous regeneration at its rostral (subcommissural organ) attachment. This type of process would require a feedback system controlling the rate of release of secretory material from the subcommissural organ.

All the previous considerations lead us to suggest, as a working hypothesis: (i) The CSF-contacting systems ex- isting along the entire length of the central canal may parti- cipate in the feedback mechanism regulating the secretory activity of the subcommissural organ. This would require that the ascending SOM-IR and UII-IR tracts originating in CSF-contacting (Reissner's fibre-contacting?) neurons establish contacts with neuronal systems innervating the subcommissural organ, such as the serotoninergic nuclei of the raphe. (ii) The "caudal serotoninergic system" might participate in the feedback mechanism by modulating the activity of local CSF-contacting systems.

Rodriguez et al. (1987) described a change in the corbo- hydrate moiety of the glycoproteins forming part of the massa caudalis in comparison to its chemical configuration in the more rostral portion of, Reissner's fiber. This fact, in addition to the increasing caliber of the fiber (probably related to a change in the mode of packing the material), might facilitate the access of antibodies to epitopes that remain unexposed in the regular thread-like configuration of the teleostean Reissner's fiber, thus leading to an in- creased AFRU-immunoreactivity of the massa caudalis. This type of structural change could be related to a process- ing associated with the suggested passage of Reissner's fiber

material into the circulation, a fact supported by light mi- croscopy (Olsson ]955), conventional electron microscopy (Sterba and Naumann 1966; Oksche 1969; Hofer et al. 1984), ultrastructural immunocytochemistry (Peruzzo et al. 1987), and the findings presented here indicating a contin- uity between the caudal end of the terminal ventricle and a blood vessel. In addition to a vascular route, some materi- al may be discharged into the surrounding connective tissue, a fact observed previously in several fish species (Olsson 1955).

In summary, the present results show the existence of a "caudal serotoninergic system" in the caudal spinal cord of coho salmon. The pattern of the 5HT-IR neurons be- longing to this system corresponds to the location of UI- UII-IR caudal secretory neurons. The distribution of 5HT- IR thin, beaded processes may indicate a regulatory role of serotonin in the function of local caudal secretory and CSF-contacting neurons. An effect on more distant targets might be mediated via a release of serotonin into the men- ingeal vessels and/or into the subarachnoid space from sero- tonin-containing nerve terminals in close contact with the external surface of the spinal cord.

Acknowledgements. The authors are grateful to pisciculture "Mares australes" for providing the specimens of coho salmon used in this study, to Dr. Karl Lederis who supplied us with UI and UII antisera, to Dr. Adolf Weindl for the SOM-As, and to C. Lizama for preparation of the schematic drawings.

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Accepted October 19, 1989