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The terminal nerve in turbot, Psetta maxima:
Journal of Chemical Neuroanatomy 24 (2002) 199 /209 www.elsevier.com/locate/jchemneu
The terminal nerve in turbot, Psetta maxima: A developmental immunocytochemical study B. Prego, M.J. Dolda´n, P. Cid, E. de Miguel Villegas * Department of Functional Biology, Laboratory of Cell Biology, University of Vigo, 36200 Vigo, Spain Received 12 November 2001; received in revised form 13 May 2002; accepted 31 May 2002
Abstract The ontogeny and organization of the terminal nerve (TN) during turbot development was studied using an antiserum to neuropeptide Y. First immunoreactive cells were detected in the olfactory placode at hatching time. At 1 day after hatching, a loose group of labeled neurons form an extracranial primordial ganglion of the TN. During the subsequent larval development, more perikarya displaying increased immunoreactivity were found along the course of the olfactory nerve. Moreover, labeled cells cross the meninx of the forebrain gathering in the olfactory bulb of larval turbot. Projections from these cells, directed both to the caudal brain and to the retina, develop when the cells become established in the olfactory bulb. The generation of immunoreactive cells in the olfactory organ extends into the metamorphic period, when a pronounced asymmetry affects the turbot morphology. At this time, the topological location of the immunoreactive cells in the TN becomes distorted. This developmental pattern was compared with those found in other teleosts and in other vertebrates. Preabsorption experiments of anti-neuropeptide Y serum with neuropeptide Y and FMRF-amide suggests that immunoreactive material observed in TN cells was not neuropeptide Y, and raises the possibility that other peptides, e.g. FMRF-amide-like peptides, exist in this neural system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: NPY; FMRF-amide; Immunocytochemistry; Flatfish; Teleost; Forebrain; Differentiation
1. Introduction The terminal nerve (TN, cranial nerve 0) is a supernumerary nerve, that has been reported in all vertebrates except agnathans (Eisthen and Northcutt, 1996). In teleosts, as in most other vertebrates, the TN is closely associated with the olfactory projection and its organization shows important particularities. Thus, cells of the TN form several discrete ganglia along the trajectory of the nerve, the caudalmost cell groups lying in an intracerebral position, inside the olfactory bulbs or the anterior telencephalon (Halpern-Sebold and Schreibman, 1983; Nozaki et al., 1985; Mu¨nz and Claas, 1987; Parhar et al., 1994, 1995; Nevitt et al., 1995; Chiba et al., 1996a,b; Chiba, 1997a,b; Pinelli et al., 2000). Fibers originating from TN ganglion cells reach different brain regions, as well as the olfactory epithelium and the
* Corresponding author. Tel.: /34-986-812-388; fax: /34-986-812556 E-mail address: [email protected] (E. de Miguel Villegas).
retina (Bartheld and Meyer, 1986; Sloan and Demski, 1987; Stell and Walker, 1987). The presence of this latter projections constitutes a portion of the retinopetal system of teleosts, and supports the frequent designation of the TN ganglion cells of teleosts as nucleus olfactoretinalis (NOR) (Mu¨nz et al., 1982; Mu¨nz and Claas, 1987; Uchiyama, 1989). Identification of gonadotropinreleasing hormone (GnRH; LHRH) in the TN of vertebrates (reviewed in Demski, 1993) has served as a useful tool to understand both the functions and origin of the TN. Thus, it has been suggested that GnRH of the TN constitutes a neuromodulatory system (Oka, 1992, 1993, 1997) involved in the regulation of sexual and reproductive behaviours (Schreibman and Margolis-Nunno, 1987; Sloan and Demski, 1987; Parhar et al., 1994). In addition, other neuractive peptides occur in the TN. Thus, the presence of FMRF-amide like immunoreactivity has been demonstrated in fishes and amphibians (Stell et al., 1984; Ekstro¨m et al., 1988; Uchiyama et al., 1988; Ostholm et al., 1990; D’Aniello et al., 1996a; Pinelli et al., 2000; Castro et al., 2001) and
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recent data indicate that NPY is also present in this neural system (Chiba et al., 1996a,b; D’Aniello et al., 1996b; Hilal et al., 1996; Subhedar et al., 1996; Chiba, 1997a; Castro et al., 1999). Concerning the origin of the TN, there is a general agreement that the TN cells arise from the olfactory placode, and migrate along the olfactory pathway. This point of view is supported by previous studies in teleosts (Chiba et al., 1994, 1996a,b; Northcutt and Muske, 1994; Parhar et al., 1995), and experimental data obtained in amphibians (Murakami et al., 1992; Northcutt and Muske, 1994), birds (Yamamoto et al., 1996) and mammals (Schwanzel-Fukuda and Pfaff, 1989; Tobet et al., 1996). In the present study, we observe a distinct labeling of the TN in a teleost, the turbot, by using a polyclonal antiserum to NPY (Diasorin) raised in rabbit. Turbot is a pleuronectiform fish (O. Percomorpha , Class Teleosts) which have an extremely complex life cycle comprising several developmental periods: embryonic, larval, metamorphic, juvenile and adult period. Turbot larvae maintain a bilateral body symmetry until the onset of the metamorphosis, whose most outstanding events are eye migration and body flattening. At the end of the metamorphosis, turbots are flat and both eyes lie on the left side of the body. Effects of metamorphosis in the nervous system of flatfishes have been correlated with the appearance of an extensive asymmetry in the olfactory system (Prasada Rao and Finger, 1984; Brin˜o´n et al., 1993), as well as with specific changes in the visual system, in which asymmetry mainly affects eye position (De Miguel Villegas et al., 1997) but not visual projections (Luckenbill-Edds and Sharma, 1977; Medina et al., 1993). The ontogeny of the turbot TN reported here shows that early developmental events affecting this neural system are largely similar to those found in vertebrates. However, the TN neurogenetic period overlaps with the metamorphic transformation. Effects of the turbot metamorphosis on the anatomical organization of the TN are described and analyzed.
2. Material and methods 2.1. Animals Turbots (Psetta maxima , previously Scophthalmus maximus ) belonging to different developmental periods were provided either by the High Technology Center (HIB) in Bergen (Norway) or by local breeders (Piscı´cola del Morrazo, S.A., Cangas; Isidro de la Cal, Meira´s-Valdovin˜o, Northwest Spain). Different criteria were used to stage turbots at various periods of their life cycle. Age of embryos was determined according to the number of hours after fertiliza-
tion (pf). Larvae were staged by age from hatching. Metamorphic turbots were classified into initial, intermediate and final stages, depending on the degree of eye migration and body flattening, while juvenile specimens were staged according to their standard length. Twenty embryos (55 /80 h pf), 50 larval (from hatching to 15 days after hatching), seven metamorphic and eight juvenile (12 /20 cm in length) specimens were used in this study. Protocols for animal experimentation were performed under the guidelines established by the Spanish Royal Decree 223/1988.
2.2. Immunohistochemical methodology and controls Animals were anaesthetized with 0.03% tricaine metanesulphonate (MS-222, Sigma, Madrid, Spain). All samples were fixed by immersion in acid-free Bouin’s fluid, dehydrated, embedded in paraffine and sectioned (7 /12 mm thick) in transverse, sagittal and horizontal planes in a rotary microtome. Serial sections were stubbed on gelatin-coated slides. The sections were dewaxed, rehydrated and washed in 0.01 M phosphate buffered saline (PBS, pH 7.2). In order to eliminate the endogenous peroxidase activity, sections were incubated with 3% H2O2 for 30 min at room temperature. Afterwards, the sections were preincubated for 1 h in 10% normal goat serum (NGS, Sigma, Madrid, Spain) in 0.5% Triton X-100 in 0.01 M PBS (pH 7.2) (PBSTX). The tissue was then incubated in 1:2000 rabbit anti-NPY (DiaSorin, Stillwater, USA) diluted in PBSTX containing 3% NGS. After several washes in PBS, subsequent steps for the avidin-biotin method included 1 h incubation with biotinylated goat-anti rabbit IgG (DAKO, Barcelona, Spain), diluted 1:100 in PBSTX with 3% NGS, and 1 h incubation with streptavidin-biotinylated horseradish peroxidase (ABC kit, DAKO, Barcelona, Spain). All incubation steps were performed at room temperature in a humid chamber, and were followed by several washes in PBS. The immunocomplex was revealed by incubating the sections with 0.05% 3,3?diaminobenzidine (DAB, Sigma, Madrid, Spain), and 0.03% H2O2 in 0.01 M PBS (pH 7.2). The reaction was allowed to occur in dark. After rinsing in PBS, sections were dehydrated and cover-slipped with DePeX. Some sections were counterstained with haematoxylin before mounting. The NPY antiserum was purchased from DiaSorin (Stillwater, USA). It did not cross-react neither with other members of the regulatory peptide family, nor with endorphin, vasoactive intestinal peptide, cholecystokinin or somatostatin. Although it has been suggested that FMRF-amide may cross-react with NPY antisera (Sheng and Cheng, 1989; Fischer et al., 1996), no data on cross-reaction of the antiserum with FMRF-amide were provided by the manufacturers.
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To test the immunocytochemical procedure, sections were incubated withouth the primary antiserum. This control resulted in no specific immunoreactive staining. To check the specificity of the antibody, alternate serial sections were collected on two series of slides. One series was processed according to the immunohistochemical procedure previously described, and the other series was incubated with primary antiserum preabsorbed either with synthetic NPY (10 mM) or with synthetic FMRF-amide (0.4 mM), both from Sigma (Barcelona, Spain). 2.3. Cell count An estimation of the mean number of TN cells during the metamorphic and juvenile period was carried out by counting nuclei of stained cells in serial sections. The Abercrombie correction factor (Abercrombie, 1946) was applied to compensate for possible double counting of nuclei in adjacent sections. The cells were counted at / 400 magnification and cell measurements were carried out with a Nikon light microscope equipped with an eyepiece micrometer at /1000 under an oil immersion objective.
3. Results In the present study, we observe a distinct labeling of the TN in a teleost, the turbot, by using a polyclonal antiserum to NPY raised in rabbit. The labeling of TN persists after preabsorption of antiserum with NPY, but was abolished in other areas of the nervous system. When the antiserum was preabsorbed with FMRFamide, the label intensity was reduced in the TN, but not in other areas of the brain. As a whole, controls performed to check the specificity of this antiserum to NPY indicate that it detects the NPY peptide in most of the brain without cross-react with FMRF-amide. By contrast, in the TN of turbot, immunostained cells with anti-NPY serum do not contain NPY, but probably a peptide that must share structural homology with the neuropeptides of the FMRF-amide family. However, analysis of the immunostaining reveals that this antiserum marks unambiguously the TN cells In posthatching turbots, the anti-NPY antibody stained different elements of the TN but no labeling could be found in embryos. As development proceeds, the distribution of immunoreactive elements becomes more complex. At hatching, the brain is very immature, and no anatomical distinction between olfactory bulbs and telencephalic hemispheres could be established. A few faintly immunostained cells were observed scattered in the olfactory placode. These cells were round-shaped and occurred preferentially at the base of the ventro-
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caudal half of the olfactory placode. Thin labeled processes from these cells entered the neuropilar area of the nearby forebrain, at the site of the developing olfactory bulb (Fig. 1A). By 1 day after hatching, immunoreactive cells in the olfactory placode became more numerous and more intensely stained. Over the next 2 days, a thin olfactory nerve connects the peripheral olfactory organ to the forebrain. Moreover, a group of labeled cells can be seen for the first time along the way of the olfactory nerve, forming a pressumptive primordial ganglion of the TN of roundshaped loosely aggregated cells (Fig. 1B). In 4 days posthatching larvae, the anlage of sessile olfactory bulbs was distinguishable in the anteroventral region of the telencephalon, and the pattern of immunoreactivity that characterizes the larval period was already established. From this age onwards, there was a progressive decrease in the number of labeled cells located in the olfactory organ. The primordial TN ganglion was present throughout the whole larval period and lay close to the anteroventral aspect of the olfactory bulbs (Fig. 1C). Ganglion cells were closely grouped, and a few large labeled cells in continuum with the cells of the ganglion penetrated the olfactory bulbs (Fig. 1D). As larval development proceeds, more labeled cells entered the forebrain forming a compact group in the rostroventral olfactory bulbs. The distance between the olfactory placode and the brain was progressively more prominent, and the course of the olfactory nerve could be observed. In 6 days posthatching larvae, a continuous strand of round or fusiform immunoreactive cells join the olfactory nerve forming a discrete cord between the olfactory epithelium and the TN ganglion (Fig. 1E). Labeled fibers arising from TN cells in the olfactory bulb were clearly observed from 9 days old larvae onwards (Fig. 1F). They coursed caudally through the ventral telencephalon to the diencephalon. Most of these fibers could be followed to the caudal hypothalamus, but a portion of them turned ventral in the preoptic region entering the optic tract and reaching the retina. These fibers formed the olfacto-retinal centrifugal pathway, and thus some NPY like-ir TN cells must be considered as retinopetal cells. The different components of the olfactory system become more conspicuous during the metamorphic period. The olfactory epithelium is now prominent and the olfactory nerve is thick. The olfactory bulbs are clearly identifiable as two symmetric solid masses at the beginning of the metamorphosis (Fig. 2), that become progressively deformed as metamorphosis proceeds. Consequently, both in late metamorphic and juvenile turbots, the forebrain becomes displaced with regard to the larval plane of bilateral symmetry, while the orientation of the rest of the brain does not change with respect to this plane. The rostral end of the left
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Fig. 1
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Fig. 2. Brightfield photomicrographs showing NPY like-ir neurons of the TN from an early metamorphic turbot. (A, B)Transverse sections through the rostral (A) and caudal (B) olfactory bulbs. Note the prominent perikarya (arrowheads) gathered into several clusters. Scale bars/40 mm.
olfactory bulb is ahead of the right one, and a slight mounting of both olfactory bulbs occurs (Figs. 3 and 5). Throughout the whole metamorphic period, occasional immunostained cells were observed in the olfactory epithelium. Strongly labeled cells appeared associated with the olfactory nerve forming either discrete strands or small clusters. Processes from these cells ran caudally towards the forebrain in the marginal zone of the olfactory nerve. In the forebrain, labeled perykaria extended caudally throughout the ventrolateral region of the olfactory bulbs, but never entered the telencephalic hemispheres. At the beginning of the metamorphosis, distribution of immunoreactive cells in the olfactory bulbs was symmetric (Fig. 2A and B), but during metamorphosis an asymmetric distribution gradually arose (Fig. 3A /F). Thus, whereas labeled cells in the right olfactory bulb distributed preferably in a more ventromedial position (Fig. 3A /C), in the left one, labeled cells always lay in
the ventrolateral margin (Fig. 3D/F). In both olfactory bulbs, immunostained cells formed three or four discrete clusters of cells. No major differences in the number of NPY like-ir cells between either sides of the TN could be detected during the metamorphosis (Table 1). Projections from TN cells are more conspicuous than in the larval period. In the later metamorphic stages, the initial course of the TN projections in the forebrain differs between the right and left side. Whereas fibers from the left side run grouped by the lateral olfactory bulb and telencephalon, those of the right side were loosely arranged through the forebrain. Immunoreactive fibers included in the olfacto-retinal centrifugal pathway lay in a marginal position in the optic nerve (Fig. 4). Within the retina these fibers ran at the limit between the inner plexiform and inner nuclear layers. In juvenile turbots, the forebrain exhibited a significant degree of asymmetry in the size and the position of the two olfactory bulbs (Fig. 5A /C). However, both
Fig. 1. Brightfield photomicrographs showing the sequence of differentiation of NPY like-ir cells of the TN during the larval period. (A) Transverse section through the forebrain in a newly hatched larva. A few NPY like-ir cells (arrowheads) can be seen in the olfactory placode. Fine fibers (arrow) arising from these cells reach the anlage of the olfactory bulbs in the forebrain. The dashed line outlines one olfactory placode. A star indicates the eye. (B) Transverse section through the forebrain in a larva 2 days after hatching. NPY like-ir cells (arrowheads) begin to migrate and aggregate near the olfactory placode. The asterisk indicates the olfactory placode. Note the presence of immunoreactive cells (arrow) in the olfactory placode. (C) Transverse section through the forebrain in a larva 4 days after hatching. NPY like-ir cells aggregate forming a primordial extracranial ganglion of the TN (arrowheads). Asterisks signal the olfactory placodes. A star indicates the pineal organ. (D) Transverse section through the forebrain in a larva 6 days after hatching. NPY like-ir cells in the developing olfactory bulb are observed (arrowheads). Thick arrows signal pigment granules. (E) Sagittal section through the rostral part of the head in a larva 6 days after hatching. Several groups of NPY like-ir cells (arrowheads) can be seen along the olfactory nerve. (F) Sagittal section through the forebrain in a larva 11 days after hatching. Note the NPY like-ir neurons both in the olfactory placode (arrow) and in the rostral olfactory bulbs (arrowhead). Projections arising from these cells course caudally to the hypothalamus (double arrows). Asterisk indicates the olfactory placode. T, telencephalon. Scale bars/20 mm in A, 10 mm in B, 20 mm in C, 15 mm in D, 20 mm in E,
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Fig. 3. Brightfield photomicrographs showing the asymmetric distribution of NPY like-ir cells in the TN of a late metamorphic turbot. The plane of the section is horizontal throughout the forebrain. (A /C) Consecutive sections of the right olfactory bulb (rO) showing several clusters of NPY likeir neurons (arrowheads) distributing throughout the olfactory nerve (A), and the right olfactory bulb (B, C). (D /F) Consecutive sections of the left olfactory bulb (lO) showing groups of NPY like-ir neurons located in a ventrolateral position (arrowheads). Scale bars/40 mm.
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olfactory bulb is opposite to the rostral cluster on the right olfactory bulb (Fig. 5A).
4. Discussion
Fig. 4. Brightfield photomicrograph showing the initial course of NPY like-ir fibers (arrowheads) in the optic nerve in a late metamorphic turbot. These fibers run preferentially by the margin of the optic nerve. Scale bar/20 mm.
olfactory bulbs exhibited an histological organization in concentric layers. In comparison to the metamorphic turbots, labeled TN-cells increased in number (Table 1) and no immunoreactivity was seen in the epithelium of the olfactory organ. Isolated immunoreactive somata were found dispersed in the olfactory nerve in a marginal location. Two distinct clusters of medially located labeled cells were observed, both rostrally and caudally, in both olfactory bulbs. These neuronal groups became more conspicuous in the right olfactory bulb than in the left one. In keeping with the topological distortion in the forebrain, not alignments of these clusters exist between the two olfactory bulbs. Occasionally, the position of the caudal cluster on the left
In this immunocytochemical study we describe the development of the TN in a teleost, the turbot, by using an antiserum that previously has been applied successfully to localize NPY-containing neurons in fishes (Subhedar et al., 1996; Castro et al., 1999). It has been shown that criteria based on the presence of neuroactive peptides in the TN are suitable to identify separately this neural system from other anatomically related olfactory projections (Demski, 1993). Thus, neuropeptides have never been located either in the olfactory pathway or in extrabulbar olfactory projections, which contain specific cytoplasmic proteins and cell-surface glycoconjugates (Hofmann and Meyer, 1991, 1995; Szabo et al., 1991; Becerra et al., 1994; Pellier and Astic, 1994; Anado´n et al., 1995; Porteros et al., 1997; Dı´az-Regueira and Anado´n, 2000). Conversely TN neurons contain specific molecular forms of GnRH and molecules related to the molluscan cardioexcitatory tetrapeptide (FMRF-amide) (Schwanzel-Fukuda et al., 1985; Wirsig and Getchell, 1986; Hofmann and Meyer, 1991; Vecino and Ekstro¨m, 1992; Kim, et al., 1995; Kyle et al., 1995; White and Meredith, 1995; Yamamoto et al., 1995; Fischer et al., 1996; Pinelli et al., 2000). More recently, NPY immunoreactivity has been extensively observed in the TN of fishes (Chiba and Honma, 1992; Chiba et al., 1996a; Subhedar et al., 1996; Chiba, 1997a; Castro et al., 1999). Apparently, an analogous immunocytochemical labeling is present in the TN of the turbot, but whereas the specificity of the reaction in most of the turbot brain is confirmed by preabsorption experiments, in the TN the staining was not affected. This result suggests that the molecule recognized by the antiserum in this neural system is not NPY. Characterization of this peptide is beyond the scope of this study. Several lines of evidence indicate that FMRFamide-like immunoreactivity in the TN of fishes is due to the presence of F8Famide and A18Famide rather FMRFamide itself (Kyle et al., 1995; Fischer et al., 1996). If we take into account that the Cterminal amino acid sequence of these peptides differs
Table 1 Comparison of mean numbers of NPY like-ir neurons (including those in the olfactory organ, TN and olfactory bulbs) between either sides of the TN in metamorphic and juvenile turbots
Initial metamorphosis Intermediate metamorphosis Final metamorphosis Juvenile turbots
Number of NPY like-ir cells in the right side of turbot TN
Number of NPY like-ir cells in the left side of turbot TN
24 25 24 29
25 24 22 30
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Fig. 5. Brightfield photomicrographs showing the distribution of NPY like-ir cells in the terminal nerve of juvenile turbots. (A) A prominent group of NPY like-ir cells (arrowhead) can be seen in the left olfactory bulb that is on advance with respect to the right one. The asterisk indicates the olfactory nerve. Note that as consequence of the distortion, the plane of the section is horizontal throughout the olfactory bulbs, and sagittal in the rest of the brain. (B, C) Horizontal (B) and transverse (C) sections through the olfactory bulbs showing the NPY like-ir groups of cells (arrowheads). lO, left olfactory bulb; rO, right olfactory bulb; ON, optic nerve; T, telencephalon. Scale bars/230 mm in A, 100 mm in B, 50 mm in C.
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from that of NPY at only one position, it is likely that the antisera used in this study visualize these peptides and/or equivalent forms. Both spatial and temporal variations in the distribution of immunoreactive neurons in the TN of developing turbot are consistent with a pattern of migration of TN cells from the olfactory placode to the forebrain. Evidence that precursors of TN cells reside initially in the olfactory placode has been extensively demonstrated in tetrapods (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989a,b; Daikoku-Ishido et al., 1990; Ronnekleiv and Resko, 1990; Norgren and Lehman, 1991; Murakami et al., 1992; D’Aniello et al., 1994; Pellier and Astic, 1994). In fishes, early precursor cells of the TN have only been identified in the olfactory placode of teleosts (Parhar et al., 1995; Chiba et al., 1996a; Pinelli et al., 2000; present results), but the accurate origin of this system in other fishes remains unclear. Thus, whereas in cyclostomes the existence of the TN nerve is questioned (Bartheld et al., 1987; Eisthen and Northcutt, 1996), both in selaceans (Chiba et al., 1996a) and sturgeons (Pinelli et al., 2000), the rudimentary ganglion of the TN constitutes the first place in which cells expressing neuropeptides were detected. This indicates that in these species either the amount of the different neuropeptides in precursor cells of the olfactory placode was too low to be detected by immunocytochemical methods, or neuropeptide expression was delayed until cells migrate out of the olfactory organ. In turbot, first immunoreactive cells in the olfactory placode appear at hatching time, whereas in other teleost species TN cells containing different neuropeptides begin to differentiate during embryonic development (Parhar et al., 1995; Chiba et al., 1996a; Parhar and Iwata, 1996; Castro et al., 1999; Pinelli et al., 2000). The apparent delay observed in the emergence of TN cells in turbot must be correlated to the slow brain development in this fish, that also affects the emergence of other neural structures (Dolda´n et al., 2000). Soon after their generation, immunoreactive cells of the turbot TN begin to migrate to the brain, reaching an intracerebral location in the olfactory bulb. As occurs in most vertebrates the sequence of events involved in the development of the TN system in turbot occurs gradually, and it is not affected by the metamorphosis. Conversely, in salmon, the appearance of TN cells in the forebrain is delayed at the time of smolt-transformation (Parhar et al., 1995; Parhar and Iwata, 1996). In larval turbot, a significant development of the caudal projections of cells of the TN towards their targets is observed in temporal coincidence with the establishment of the cells inside the olfactory bulb, being apparently symmetrical and equal in both sides of the brain. However, the TN ontogeny is not completed during the larval period, since labeled neurons are observed in the olfactory organ until the metamorpho-
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sis. In fact, both cell migration and localization of TN cells in the olfactory bulb are strongly affected by the distortion suffered in the forebrain, although no clear differences in the number of immunoreactive cells were apparent in the TN at both sides of the brain. Additionally, TN axons proceeded to the telencephalic hemispheres by different routes. As a whole, variations of the TN components of the right and left side observed in this study, appear to be topological adaptations to the brain deformation. In the flatfish Pseudopleuronectes americanus , the olfactory system is strongly asymmetric (Prasada Rao and Finger, 1984). In this case, however, differences in the primary and secondary olfactory projections appear together with uneven extensions of the main target territories (the olfactory bulbs and the telencephalic hemispheres, respectively). That it is not the case for the TN, formed by a roughly similar contingent of neurons in both sides of the brain, that maintain connections with symmetric areas of the brain. Hence, it is conceivable that asymmetry in this neural system, if present, is significantly reduced.
Acknowledgements This work was supported by a grant from the University of Vigo UVIGO99 (64102C006). We thank Dr R. Anado´n, Dr J.M. Cerda´-Reverter and the anonymous reviewers for helpful comments on the manuscript, and we thank I. Emmett and A. Gonza´lez for the stylistic correction of the manuscript. Turbots were supplied by Piscı´cola del Morrazo (Nerga, Spain), Isidro de la Cal (Meira´s-Valdovin˜o, Spain), and the High Technology Center (Bergen, Norway).
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Castro, A., Becerra, M., Manso, M.J., Anado´n, A., 1999. Development of immunoreactivity to neuropeptide Y in the brain of brown trout (Salmo trutta fario ). J. Comp. Neurol. 414, 13 /32. Castro, A., Becerra, M., Anadon, R., Manso, M.J., 2001. Distribution and development of FMRFamide-like immunoreactive neuronal systems in the brain of the brown trout, Salmo trutta fario . J. Comp. Neurol. 440, 43 /64. Chiba, A., Honma, Y., 1992. Distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the cloudy dogfish, Scyliorhinus torazame . Cell Tissue Res. 268, 453 /461. Chiba, A., Oka, S., Honma, Y., 1994. Ontogenetic development of gonadotropin-releasing hormone-like immunoreactive neurons in the brain of the chum salmon, Oncorhynchus keta . Neurosci. Lett. 178, 51 /54. Chiba, A., Oka, S., Honma, Y., 1996a. Ontogenetic changes in neuropeptide Y-like-immunoreactivity in the terminal nerve of chum salmon and the cloudy dogfish, with special reference to colocalization with gonadotropin-releasing hormone-immunoreactivity. Neurosci. Lett. 213, 49 /52. Chiba, A., Sohn, Y.C., Honma, Y., 1996b. Distribution of neuropeptide Y and gonadotropin-releasing hormone immunoreactivities in the brain and hypophysis of the ayu, Plecoglossus altivelis (Teleostei). Arch. Histol. Cytol. 59, 137 /148. Chiba, A., 1997a. Colocalization of gonadotropin-releasing hormone (GnRH)-, Neuropeptide Y (NPY)-, and molluscan cardioexcitatory tetrapeptide (FMRFamide)-like immunoreactivities in the ganglion cells of the terminal nerve of the masu salmon. Fisheries Sci. 63, 153 /154. Chiba, A., 1997b. Distribution of neuropeptide Y-like immunoreactivity in the brain of the bichir, Polypterus senegalus , with special regard to the terminal nerve. Cell Tissue Res. 289, 275 /284. D’Aniello, B., Masucci, M., di Meglio, M., Iela, L., Rastogi, R.K., 1994. Immunohistochemical localization of GnRH in the crested newt (Triturus carnifex ) brain and terminal nerve: a developmental study. J. Neuroendocrinol. 6, 167 /172. D’Aniello, B., Fiorentino, M., Pinelli, C., di Meglio, M., Vallarino, M., Rastogi, R.K., 1996a. Distribution of FMRFamide-like immunoreactivity in the brain and pituitary of Rana esculenta during development. Brain Res. Dev. Brain Res. 95, 194 /204. D’Aniello, B., Vallarino, M., Pinelli, C., Fiorentino, M., Rastogi, R.K., 1996b. Neuropeptide Y: localization in the brain and pituitary of the developing frog (Rana esculenta ). Cell Tissue Res. 285, 253 /259. Daikoku-Ishido, H., Okamura, Y., Yanaihara, N., Daikoku, S., 1990. Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: in vivo and in transplantation studies. Dev. Biol. 140, 374 /387. De Miguel Villegas, E., Dolda´n Dans, M.J., Paz-Andrade Castillo, C., Anado´n Alvarez, R., 1997. Development of the eye in the turbot Psetta maxima (Teleosti) from hatching through metamorphosis. J. Morphol. 233, 31 /42. Demski, L.S., 1993. Terminal nerve complex. Acta Anat. 148, 81 /95. Dı´az-Regueira, S., Anado´n, R., 2000. Calretinin expression in specific neuronal systems in the brain of an advanced teleost, the grey mullet (Chelon labrosus ). J. Comp. Neurol. 426, 81 /105. Dolda´n, M.J., Prego, B., Holmqvist, B., Helvik, J.V., de Miguel, E., 2000. Emergence of axonal tracts in the developing brain of the turbot (Psetta maxima ). Brain Behav. Evol. 56, 300 /309. Eisthen, H.L., Northcutt, R.G., 1996. Silver lampreys (Ichthyomyzon unicuspis ) lack a gonadotropin-releasing hormone- and FMRFamide-immunoreactive terminal nerve. J. Comp. Neurol. 370, 159 / 172. Ekstro¨m, P., Honkanen, T., Ebbesson, S.O.E., 1988. FMRFamide-like immunoreactive neurons of the nervus terminalis of teleosts innervate both retina and pineal organ. Brain Res. 460, 68 /75.
Fischer, A.J., Reisch, H.M., Hyle, A.L., Stell, W.K., 1996. Characterization of the RFamide-like neuropeptides in the nervus terminalis of the goldfish (Carassius auratus ). Regul. Pept. 62, 73 /87. Halpern-Sebold, L.R., Schreibman, M., 1983. Ontogeny of centers containing luteinizing hormone-releasing hormone in the brain of platyfish (Xiphophorus maculatus ) as determined by immunocytochemistry. Cell Tissue Res. 229, 75 /84. Hilal, E.M., Chen, J.H., Silverman, A.J., 1996. Joint migration of gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY) neurons from olfactory placode to central nervous system. J. Neurobiol. 31, 487 /502. Hofmann, M.H., Meyer, D.L., 1991. Subdivisions of the terminal nerve in Xenopus laevis . J. Exp. Zool. 259, 324 /329. Hofmann, M.H., Meyer, D.L., 1995. The extrabulbar olfactory pathway: primary olfactory fibers bypassing the olfactory bulb in bony fishes? Brain Behav. Evol. 46, 378 /388. Kim, M.-E., Oka, Y., Amano, M., Kobayashi, M., Okuzawa, K., Hasegawa, Y., Kawashima, S., Suzuki, Y., Aida, K., 1995. Immunocytochemical localization of sGnRH and cGnRH-II in the brain of goldfish, Carassius auratus . J. Comp. Neurol. 356, 72 / 82. Kyle, A.L., Luo, B., Magnus, T.H., Stell, W.K., 1995. Substance P-, F8Famide-, and A18Famide-like immunoreactivity in the nervus terminalis and retina of the goldfish Carassius auratus . Cell Tissue Res. 280, 605 /615. Luckenbill-Edds, L., Sharma, S.C., 1977. Retinotectal projection of the adult winter flounder (Pseudopleuronectes americanus ). J. Comp. Neurol. 173, 307 /318. Medina, M., Reperant, J., Ward, R., Rio, J.-P., Lemire, M., 1993. The primary visual system of flatfish: an evolutionary perspective. Anat. Embryol. 187, 167 /191. Mu¨nz, H., Claas, B., Stumpf, W.E., Jennes, L., 1982. Centrifugal innervation of the retina by luteinizing hormone releasing homone (LHRH)-immunoreactive telencephalic neurons in teleostean fishes. Cell Tissue Res. 222, 313 /323. Mu¨nz, H., Claas, B., 1987. The terminal nerve and its development in the teleost fishes. Ann. N.Y. Acad. Sci. 519, 50 /59. Murakami, S., Kikuyama, S., Arai, Y., 1992. The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster ): the effect of olfactory placode ablation. Cell Tissue Res. 269, 21 /27. Nevitt, G.A., Grober, M.S., Marchaterre, M.A., Bass, A.H., 1995. GnRH-like immunoreactivity in the peripheral olfactory system and forebrain of atlantic salmon (Salmo salar ): a reassessment at multiple life history stages. Brain Behav. Evol. 45, 350 /358. Norgren, R.B., Lehman, M.N., 1991. Neurons that migrate from the olfactory epithelium in the chick express luteinizing hormonereleasing hormone. Endocrinology 128, 1676 /1678. Northcutt, R.G., Muske, L.E., 1994. Multiple embryonic origins of gonadotropin-releasing hormone (GnRH) immunoreactive neurons. Dev. Brain Res. 78, 279 /290. Nozaki, M., Fujita, I., Saito, N., Tsukahara, T., Kobayashi, H., Ueda, K., Oshima, K., 1985. Distribution of LHRH-like immunoreactivity in the brain of the japanese eel (Anguilla japonica ) with special reference to the nervus terminalis. Zool. Sci. 2, 537 /547. Oka, Y., 1992. Gonadotropin-releasing hormone (GnRH) cells of the terminal nerve as a model neuromodulator system. Neurosci. Lett. 142, 119 /122. Oka, Y., 1993. Gonadotropin-releasing hormone (GnRH)-immunoreactive terminal nerve cells have intrinsic rythmicity and project widely in the brain. J. Neurosci. 13, 2161 /2176. Oka, Y., 1997. GnRH neuronal system of fish brain as a model system for the study of peptidergic neuromodulation. In: Parhar, I.S., Sakuma, Y. (Eds.), GnRH Neurons: Gene to Behavior. Brain Shuppan, Tokyo, pp. 245 /276. Ostholm, T., Ekstro¨m, P., Ebbesson, S.O.E., 1990. Distribution of FMRFamide-like immunoreactivity in the brain, retina and nervus
B. Prego et al. / Journal of Chemical Neuroanatomy 24 (2002) 199 /209 terminalis of the sockeye salmon parr, Oncorhynchus nerka . Cell Tissue Res. 261, 403 /418. Parhar, I.S., Koibuchi, N., Sakai, M., Iwata, M., Yamaoka, S., 1994. Gonadotropin-releasing hormone (GnRH): expression during salmon migration. Neurosci. Lett. 172, 15 /18. Parhar, I.S., Iwata, M., Pfaff, D.W., Schwanzel-Fukuda, M., 1995. Embryonic development of gonadotropin-releasing hormone neurons in the sockeye salmon. J. Comp. Neurol. 362, 256 /270. Parhar, I.S., Iwata, M., 1996. Intracerebral expression of gonadotropin-releasing hormone and growth hormone-releasing hormone is delayed until smoltification in the salmon. Neurosci. Res. 26, 299 / 308. Pellier, V., Astic, L., 1994. Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode. Cell Tissue Res. 275, 587 /598. Pinelli, C., D’Aniello, B., Sordino, P., Meyer, D.L., Fiorentino, M., Rastogi, R.K., 2000. Comparative immunocytochemical study of FMRFamide neuronal system in the brain of Danio rerio and Acipenser ruthenus during development. Dev. Brain Res. 119, 195 / 208. Porteros, A., Are´valo, R., Weruaga, E., Crespo, C., Brin˜o´n, J.G., Alonso, J.R., Aijo´n, J., 1997. Calretinin immunoreactivity in the developing olfactory system of the rainbow trout. Dev. Brain Res. 100, 101 /109. Prasada Rao, P.D., Finger, T.E., 1984. Asymmetry of the olfactory system in the brain of the winter flounder Pseudopleuronectes americanus . J. Comp. Neurol. 225, 492 /510. Ronnekleiv, O.K., Resko, J.A., 1990. Ontogeny of gonadotropinreleasing hormone-containing neurons in early fetal development of rhesus macaques. Endocrinology 126, 498 /511. Schreibman, M.P., Margolis-Nunno, H., 1987. Reproductive biology of the terminal nerve (nucleus olfactoretinalis) and other LHRH pathways in teleost fishes. Ann. N.Y. Acad. Sci. 519, 60 /68. Schwanzel-Fukuda, M., Morrell, J.I., Pfaff, D.W., 1985. Ontogenesis of neurons producing luteinizing hormone-releasing hormone (LHRH) in the nervus terminalis of the rat. J. Comp. Neurol. 238, 348 /364. Schwanzel-Fukuda, M., Pfaff, D.W., 1989. Origin of luteinizing hormone-releasing hormone neurons. Nature 338, 161 /164. Sheng, C.L., Cheng, J.T., 1989. Cross-reaction of FMRFamide with a neuropeptide Y (NPY) antiserum: histological and radioimmunological evidence. Biomed. Ref. 10, 517 /520. Sloan, H.E., Demski, L.S., 1987. Studies on the terminal nerve and its central connections in goldfish. Ann. N.Y. Acad. Sci. 519, 421 / 432. Stell, W.K., Walker, S.E., Chohan, K.S., Ball, A.K., 1984. The goldfish nervus terminalis: a luteinizing hormone-releasing hor-
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mone and molluscan cardioexcitatory peptide immunoreactive olfactoretinal pathway. Proc. Natl. Acad. Sci. USA 81, 940 /944. Stell, W.K., Walker, S.E., 1987. Functional-anatomical studies on the terminal nerve projection to the retina of bony fishes. Ann. N.Y. Acad. Sci. 519, 80 /96. Subhedar, N., Cerda´, J., Wallace, R.A., 1996. Neuropeptide Y in the forebrain and retina of the killifish, Fundulus heteroclitus . Cell Tissue Res. 283, 313 /323. Szabo, T., Bla¨hser, S., Denizot, J.-P., Ravaille-Ve´ron, M., 1991. Projection olfactive primaire extrabulbaire chez certaines poissons te´le´oste´ens. C. R. Acad. Sci. Paris 312, 555 /560. Tobet, S.A., Hanna, I.K., Schwarting, G.A., 1996. Migration of neurons containing gonadotropin releasing hormone (GnRH) in slices from embryonic nasal compartment and forebrain. Dev. Brain Res. 97, 287 /292. Uchiyama, H., Reh, T.A., Stell, W.K., 1988. Immunocytochemical and morphological evidence for a retinopetal projection in anuran amphibians. J. Comp. Neurol. 274, 48 /59. Uchiyama, H., 1989. Centrifugal pathways to the retina: influence of the optic tectum. Vis. Neurosci. 3, 183 /206. Vecino, E., Ekstro¨m, P., 1992. Colocalization of neuropeptide Y (NPY)-like and FMRFamide-like immunoreactivities in the brain of the Atlantic salmon (Salmo salar ). Cell Tissue Res. 270, 435 / 442. White, J., Meredith, M., 1995. Nervus terminalis ganglion of the bonnethead shark (Sphyrna tiburo ): evidence for cholinergic and catecholaminergic influence on two cell types distinguished by peptide immunocytochemistry. J. Comp. Neurol. 351, 385 /403. Wirsig, C.R., Getchell, T.V., 1986. Amphibian terminal nerve: distribution revealed by LHRH and AChE markers. Brain Res. 385, 10 /21. Wray, S., Grant, P., Gainer, H., 1989a. Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc. Natl. Acad. Sci. USA 86, 8132 /8136. Wray, S., Nieburgs, A., Elkabes, S., 1989b. Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev. Brain Res. 46, 309 /318. Yamamoto, N., Yoshitaka, O., Amano, M., Aida, K., Hasegawa, Y., Kawashima, S., 1995. Multiple gonadotropin-releasing hormone (GnRH)-immunoreactive systems in the brain of the dwarf gourami, Colisa lalia : immunohistochemistry and radioimmunoassay. J. Comp. Neurol. 355, 354 /368. Yamamoto, N., Uchiyama, H., Ohki-Hamazaki, H., Tanaka, H., Ito, H., 1996. Migration of GnRH-immunoreactive neurons from the olfactory placode to the brain: a study using avian embryonic chimeras. Dev. Brain Res. 95, 234 /244.
The terminal nerve in turbot, Psetta maxima: A developmental immunocytochemical study B. Prego, M.J. Dolda´n, P. Cid, E. de Miguel Villegas * Department of Functional Biology, Laboratory of Cell Biology, University of Vigo, 36200 Vigo, Spain Received 12 November 2001; received in revised form 13 May 2002; accepted 31 May 2002
Abstract The ontogeny and organization of the terminal nerve (TN) during turbot development was studied using an antiserum to neuropeptide Y. First immunoreactive cells were detected in the olfactory placode at hatching time. At 1 day after hatching, a loose group of labeled neurons form an extracranial primordial ganglion of the TN. During the subsequent larval development, more perikarya displaying increased immunoreactivity were found along the course of the olfactory nerve. Moreover, labeled cells cross the meninx of the forebrain gathering in the olfactory bulb of larval turbot. Projections from these cells, directed both to the caudal brain and to the retina, develop when the cells become established in the olfactory bulb. The generation of immunoreactive cells in the olfactory organ extends into the metamorphic period, when a pronounced asymmetry affects the turbot morphology. At this time, the topological location of the immunoreactive cells in the TN becomes distorted. This developmental pattern was compared with those found in other teleosts and in other vertebrates. Preabsorption experiments of anti-neuropeptide Y serum with neuropeptide Y and FMRF-amide suggests that immunoreactive material observed in TN cells was not neuropeptide Y, and raises the possibility that other peptides, e.g. FMRF-amide-like peptides, exist in this neural system. # 2002 Elsevier Science B.V. All rights reserved. Keywords: NPY; FMRF-amide; Immunocytochemistry; Flatfish; Teleost; Forebrain; Differentiation
1. Introduction The terminal nerve (TN, cranial nerve 0) is a supernumerary nerve, that has been reported in all vertebrates except agnathans (Eisthen and Northcutt, 1996). In teleosts, as in most other vertebrates, the TN is closely associated with the olfactory projection and its organization shows important particularities. Thus, cells of the TN form several discrete ganglia along the trajectory of the nerve, the caudalmost cell groups lying in an intracerebral position, inside the olfactory bulbs or the anterior telencephalon (Halpern-Sebold and Schreibman, 1983; Nozaki et al., 1985; Mu¨nz and Claas, 1987; Parhar et al., 1994, 1995; Nevitt et al., 1995; Chiba et al., 1996a,b; Chiba, 1997a,b; Pinelli et al., 2000). Fibers originating from TN ganglion cells reach different brain regions, as well as the olfactory epithelium and the
* Corresponding author. Tel.: /34-986-812-388; fax: /34-986-812556 E-mail address: [email protected] (E. de Miguel Villegas).
retina (Bartheld and Meyer, 1986; Sloan and Demski, 1987; Stell and Walker, 1987). The presence of this latter projections constitutes a portion of the retinopetal system of teleosts, and supports the frequent designation of the TN ganglion cells of teleosts as nucleus olfactoretinalis (NOR) (Mu¨nz et al., 1982; Mu¨nz and Claas, 1987; Uchiyama, 1989). Identification of gonadotropinreleasing hormone (GnRH; LHRH) in the TN of vertebrates (reviewed in Demski, 1993) has served as a useful tool to understand both the functions and origin of the TN. Thus, it has been suggested that GnRH of the TN constitutes a neuromodulatory system (Oka, 1992, 1993, 1997) involved in the regulation of sexual and reproductive behaviours (Schreibman and Margolis-Nunno, 1987; Sloan and Demski, 1987; Parhar et al., 1994). In addition, other neuractive peptides occur in the TN. Thus, the presence of FMRF-amide like immunoreactivity has been demonstrated in fishes and amphibians (Stell et al., 1984; Ekstro¨m et al., 1988; Uchiyama et al., 1988; Ostholm et al., 1990; D’Aniello et al., 1996a; Pinelli et al., 2000; Castro et al., 2001) and
0891-0618/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 1 - 0 6 1 8 ( 0 2 ) 0 0 0 5 7 - 1
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recent data indicate that NPY is also present in this neural system (Chiba et al., 1996a,b; D’Aniello et al., 1996b; Hilal et al., 1996; Subhedar et al., 1996; Chiba, 1997a; Castro et al., 1999). Concerning the origin of the TN, there is a general agreement that the TN cells arise from the olfactory placode, and migrate along the olfactory pathway. This point of view is supported by previous studies in teleosts (Chiba et al., 1994, 1996a,b; Northcutt and Muske, 1994; Parhar et al., 1995), and experimental data obtained in amphibians (Murakami et al., 1992; Northcutt and Muske, 1994), birds (Yamamoto et al., 1996) and mammals (Schwanzel-Fukuda and Pfaff, 1989; Tobet et al., 1996). In the present study, we observe a distinct labeling of the TN in a teleost, the turbot, by using a polyclonal antiserum to NPY (Diasorin) raised in rabbit. Turbot is a pleuronectiform fish (O. Percomorpha , Class Teleosts) which have an extremely complex life cycle comprising several developmental periods: embryonic, larval, metamorphic, juvenile and adult period. Turbot larvae maintain a bilateral body symmetry until the onset of the metamorphosis, whose most outstanding events are eye migration and body flattening. At the end of the metamorphosis, turbots are flat and both eyes lie on the left side of the body. Effects of metamorphosis in the nervous system of flatfishes have been correlated with the appearance of an extensive asymmetry in the olfactory system (Prasada Rao and Finger, 1984; Brin˜o´n et al., 1993), as well as with specific changes in the visual system, in which asymmetry mainly affects eye position (De Miguel Villegas et al., 1997) but not visual projections (Luckenbill-Edds and Sharma, 1977; Medina et al., 1993). The ontogeny of the turbot TN reported here shows that early developmental events affecting this neural system are largely similar to those found in vertebrates. However, the TN neurogenetic period overlaps with the metamorphic transformation. Effects of the turbot metamorphosis on the anatomical organization of the TN are described and analyzed.
2. Material and methods 2.1. Animals Turbots (Psetta maxima , previously Scophthalmus maximus ) belonging to different developmental periods were provided either by the High Technology Center (HIB) in Bergen (Norway) or by local breeders (Piscı´cola del Morrazo, S.A., Cangas; Isidro de la Cal, Meira´s-Valdovin˜o, Northwest Spain). Different criteria were used to stage turbots at various periods of their life cycle. Age of embryos was determined according to the number of hours after fertiliza-
tion (pf). Larvae were staged by age from hatching. Metamorphic turbots were classified into initial, intermediate and final stages, depending on the degree of eye migration and body flattening, while juvenile specimens were staged according to their standard length. Twenty embryos (55 /80 h pf), 50 larval (from hatching to 15 days after hatching), seven metamorphic and eight juvenile (12 /20 cm in length) specimens were used in this study. Protocols for animal experimentation were performed under the guidelines established by the Spanish Royal Decree 223/1988.
2.2. Immunohistochemical methodology and controls Animals were anaesthetized with 0.03% tricaine metanesulphonate (MS-222, Sigma, Madrid, Spain). All samples were fixed by immersion in acid-free Bouin’s fluid, dehydrated, embedded in paraffine and sectioned (7 /12 mm thick) in transverse, sagittal and horizontal planes in a rotary microtome. Serial sections were stubbed on gelatin-coated slides. The sections were dewaxed, rehydrated and washed in 0.01 M phosphate buffered saline (PBS, pH 7.2). In order to eliminate the endogenous peroxidase activity, sections were incubated with 3% H2O2 for 30 min at room temperature. Afterwards, the sections were preincubated for 1 h in 10% normal goat serum (NGS, Sigma, Madrid, Spain) in 0.5% Triton X-100 in 0.01 M PBS (pH 7.2) (PBSTX). The tissue was then incubated in 1:2000 rabbit anti-NPY (DiaSorin, Stillwater, USA) diluted in PBSTX containing 3% NGS. After several washes in PBS, subsequent steps for the avidin-biotin method included 1 h incubation with biotinylated goat-anti rabbit IgG (DAKO, Barcelona, Spain), diluted 1:100 in PBSTX with 3% NGS, and 1 h incubation with streptavidin-biotinylated horseradish peroxidase (ABC kit, DAKO, Barcelona, Spain). All incubation steps were performed at room temperature in a humid chamber, and were followed by several washes in PBS. The immunocomplex was revealed by incubating the sections with 0.05% 3,3?diaminobenzidine (DAB, Sigma, Madrid, Spain), and 0.03% H2O2 in 0.01 M PBS (pH 7.2). The reaction was allowed to occur in dark. After rinsing in PBS, sections were dehydrated and cover-slipped with DePeX. Some sections were counterstained with haematoxylin before mounting. The NPY antiserum was purchased from DiaSorin (Stillwater, USA). It did not cross-react neither with other members of the regulatory peptide family, nor with endorphin, vasoactive intestinal peptide, cholecystokinin or somatostatin. Although it has been suggested that FMRF-amide may cross-react with NPY antisera (Sheng and Cheng, 1989; Fischer et al., 1996), no data on cross-reaction of the antiserum with FMRF-amide were provided by the manufacturers.
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To test the immunocytochemical procedure, sections were incubated withouth the primary antiserum. This control resulted in no specific immunoreactive staining. To check the specificity of the antibody, alternate serial sections were collected on two series of slides. One series was processed according to the immunohistochemical procedure previously described, and the other series was incubated with primary antiserum preabsorbed either with synthetic NPY (10 mM) or with synthetic FMRF-amide (0.4 mM), both from Sigma (Barcelona, Spain). 2.3. Cell count An estimation of the mean number of TN cells during the metamorphic and juvenile period was carried out by counting nuclei of stained cells in serial sections. The Abercrombie correction factor (Abercrombie, 1946) was applied to compensate for possible double counting of nuclei in adjacent sections. The cells were counted at / 400 magnification and cell measurements were carried out with a Nikon light microscope equipped with an eyepiece micrometer at /1000 under an oil immersion objective.
3. Results In the present study, we observe a distinct labeling of the TN in a teleost, the turbot, by using a polyclonal antiserum to NPY raised in rabbit. The labeling of TN persists after preabsorption of antiserum with NPY, but was abolished in other areas of the nervous system. When the antiserum was preabsorbed with FMRFamide, the label intensity was reduced in the TN, but not in other areas of the brain. As a whole, controls performed to check the specificity of this antiserum to NPY indicate that it detects the NPY peptide in most of the brain without cross-react with FMRF-amide. By contrast, in the TN of turbot, immunostained cells with anti-NPY serum do not contain NPY, but probably a peptide that must share structural homology with the neuropeptides of the FMRF-amide family. However, analysis of the immunostaining reveals that this antiserum marks unambiguously the TN cells In posthatching turbots, the anti-NPY antibody stained different elements of the TN but no labeling could be found in embryos. As development proceeds, the distribution of immunoreactive elements becomes more complex. At hatching, the brain is very immature, and no anatomical distinction between olfactory bulbs and telencephalic hemispheres could be established. A few faintly immunostained cells were observed scattered in the olfactory placode. These cells were round-shaped and occurred preferentially at the base of the ventro-
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caudal half of the olfactory placode. Thin labeled processes from these cells entered the neuropilar area of the nearby forebrain, at the site of the developing olfactory bulb (Fig. 1A). By 1 day after hatching, immunoreactive cells in the olfactory placode became more numerous and more intensely stained. Over the next 2 days, a thin olfactory nerve connects the peripheral olfactory organ to the forebrain. Moreover, a group of labeled cells can be seen for the first time along the way of the olfactory nerve, forming a pressumptive primordial ganglion of the TN of roundshaped loosely aggregated cells (Fig. 1B). In 4 days posthatching larvae, the anlage of sessile olfactory bulbs was distinguishable in the anteroventral region of the telencephalon, and the pattern of immunoreactivity that characterizes the larval period was already established. From this age onwards, there was a progressive decrease in the number of labeled cells located in the olfactory organ. The primordial TN ganglion was present throughout the whole larval period and lay close to the anteroventral aspect of the olfactory bulbs (Fig. 1C). Ganglion cells were closely grouped, and a few large labeled cells in continuum with the cells of the ganglion penetrated the olfactory bulbs (Fig. 1D). As larval development proceeds, more labeled cells entered the forebrain forming a compact group in the rostroventral olfactory bulbs. The distance between the olfactory placode and the brain was progressively more prominent, and the course of the olfactory nerve could be observed. In 6 days posthatching larvae, a continuous strand of round or fusiform immunoreactive cells join the olfactory nerve forming a discrete cord between the olfactory epithelium and the TN ganglion (Fig. 1E). Labeled fibers arising from TN cells in the olfactory bulb were clearly observed from 9 days old larvae onwards (Fig. 1F). They coursed caudally through the ventral telencephalon to the diencephalon. Most of these fibers could be followed to the caudal hypothalamus, but a portion of them turned ventral in the preoptic region entering the optic tract and reaching the retina. These fibers formed the olfacto-retinal centrifugal pathway, and thus some NPY like-ir TN cells must be considered as retinopetal cells. The different components of the olfactory system become more conspicuous during the metamorphic period. The olfactory epithelium is now prominent and the olfactory nerve is thick. The olfactory bulbs are clearly identifiable as two symmetric solid masses at the beginning of the metamorphosis (Fig. 2), that become progressively deformed as metamorphosis proceeds. Consequently, both in late metamorphic and juvenile turbots, the forebrain becomes displaced with regard to the larval plane of bilateral symmetry, while the orientation of the rest of the brain does not change with respect to this plane. The rostral end of the left
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Fig. 1
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Fig. 2. Brightfield photomicrographs showing NPY like-ir neurons of the TN from an early metamorphic turbot. (A, B)Transverse sections through the rostral (A) and caudal (B) olfactory bulbs. Note the prominent perikarya (arrowheads) gathered into several clusters. Scale bars/40 mm.
olfactory bulb is ahead of the right one, and a slight mounting of both olfactory bulbs occurs (Figs. 3 and 5). Throughout the whole metamorphic period, occasional immunostained cells were observed in the olfactory epithelium. Strongly labeled cells appeared associated with the olfactory nerve forming either discrete strands or small clusters. Processes from these cells ran caudally towards the forebrain in the marginal zone of the olfactory nerve. In the forebrain, labeled perykaria extended caudally throughout the ventrolateral region of the olfactory bulbs, but never entered the telencephalic hemispheres. At the beginning of the metamorphosis, distribution of immunoreactive cells in the olfactory bulbs was symmetric (Fig. 2A and B), but during metamorphosis an asymmetric distribution gradually arose (Fig. 3A /F). Thus, whereas labeled cells in the right olfactory bulb distributed preferably in a more ventromedial position (Fig. 3A /C), in the left one, labeled cells always lay in
the ventrolateral margin (Fig. 3D/F). In both olfactory bulbs, immunostained cells formed three or four discrete clusters of cells. No major differences in the number of NPY like-ir cells between either sides of the TN could be detected during the metamorphosis (Table 1). Projections from TN cells are more conspicuous than in the larval period. In the later metamorphic stages, the initial course of the TN projections in the forebrain differs between the right and left side. Whereas fibers from the left side run grouped by the lateral olfactory bulb and telencephalon, those of the right side were loosely arranged through the forebrain. Immunoreactive fibers included in the olfacto-retinal centrifugal pathway lay in a marginal position in the optic nerve (Fig. 4). Within the retina these fibers ran at the limit between the inner plexiform and inner nuclear layers. In juvenile turbots, the forebrain exhibited a significant degree of asymmetry in the size and the position of the two olfactory bulbs (Fig. 5A /C). However, both
Fig. 1. Brightfield photomicrographs showing the sequence of differentiation of NPY like-ir cells of the TN during the larval period. (A) Transverse section through the forebrain in a newly hatched larva. A few NPY like-ir cells (arrowheads) can be seen in the olfactory placode. Fine fibers (arrow) arising from these cells reach the anlage of the olfactory bulbs in the forebrain. The dashed line outlines one olfactory placode. A star indicates the eye. (B) Transverse section through the forebrain in a larva 2 days after hatching. NPY like-ir cells (arrowheads) begin to migrate and aggregate near the olfactory placode. The asterisk indicates the olfactory placode. Note the presence of immunoreactive cells (arrow) in the olfactory placode. (C) Transverse section through the forebrain in a larva 4 days after hatching. NPY like-ir cells aggregate forming a primordial extracranial ganglion of the TN (arrowheads). Asterisks signal the olfactory placodes. A star indicates the pineal organ. (D) Transverse section through the forebrain in a larva 6 days after hatching. NPY like-ir cells in the developing olfactory bulb are observed (arrowheads). Thick arrows signal pigment granules. (E) Sagittal section through the rostral part of the head in a larva 6 days after hatching. Several groups of NPY like-ir cells (arrowheads) can be seen along the olfactory nerve. (F) Sagittal section through the forebrain in a larva 11 days after hatching. Note the NPY like-ir neurons both in the olfactory placode (arrow) and in the rostral olfactory bulbs (arrowhead). Projections arising from these cells course caudally to the hypothalamus (double arrows). Asterisk indicates the olfactory placode. T, telencephalon. Scale bars/20 mm in A, 10 mm in B, 20 mm in C, 15 mm in D, 20 mm in E,
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Fig. 3. Brightfield photomicrographs showing the asymmetric distribution of NPY like-ir cells in the TN of a late metamorphic turbot. The plane of the section is horizontal throughout the forebrain. (A /C) Consecutive sections of the right olfactory bulb (rO) showing several clusters of NPY likeir neurons (arrowheads) distributing throughout the olfactory nerve (A), and the right olfactory bulb (B, C). (D /F) Consecutive sections of the left olfactory bulb (lO) showing groups of NPY like-ir neurons located in a ventrolateral position (arrowheads). Scale bars/40 mm.
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olfactory bulb is opposite to the rostral cluster on the right olfactory bulb (Fig. 5A).
4. Discussion
Fig. 4. Brightfield photomicrograph showing the initial course of NPY like-ir fibers (arrowheads) in the optic nerve in a late metamorphic turbot. These fibers run preferentially by the margin of the optic nerve. Scale bar/20 mm.
olfactory bulbs exhibited an histological organization in concentric layers. In comparison to the metamorphic turbots, labeled TN-cells increased in number (Table 1) and no immunoreactivity was seen in the epithelium of the olfactory organ. Isolated immunoreactive somata were found dispersed in the olfactory nerve in a marginal location. Two distinct clusters of medially located labeled cells were observed, both rostrally and caudally, in both olfactory bulbs. These neuronal groups became more conspicuous in the right olfactory bulb than in the left one. In keeping with the topological distortion in the forebrain, not alignments of these clusters exist between the two olfactory bulbs. Occasionally, the position of the caudal cluster on the left
In this immunocytochemical study we describe the development of the TN in a teleost, the turbot, by using an antiserum that previously has been applied successfully to localize NPY-containing neurons in fishes (Subhedar et al., 1996; Castro et al., 1999). It has been shown that criteria based on the presence of neuroactive peptides in the TN are suitable to identify separately this neural system from other anatomically related olfactory projections (Demski, 1993). Thus, neuropeptides have never been located either in the olfactory pathway or in extrabulbar olfactory projections, which contain specific cytoplasmic proteins and cell-surface glycoconjugates (Hofmann and Meyer, 1991, 1995; Szabo et al., 1991; Becerra et al., 1994; Pellier and Astic, 1994; Anado´n et al., 1995; Porteros et al., 1997; Dı´az-Regueira and Anado´n, 2000). Conversely TN neurons contain specific molecular forms of GnRH and molecules related to the molluscan cardioexcitatory tetrapeptide (FMRF-amide) (Schwanzel-Fukuda et al., 1985; Wirsig and Getchell, 1986; Hofmann and Meyer, 1991; Vecino and Ekstro¨m, 1992; Kim, et al., 1995; Kyle et al., 1995; White and Meredith, 1995; Yamamoto et al., 1995; Fischer et al., 1996; Pinelli et al., 2000). More recently, NPY immunoreactivity has been extensively observed in the TN of fishes (Chiba and Honma, 1992; Chiba et al., 1996a; Subhedar et al., 1996; Chiba, 1997a; Castro et al., 1999). Apparently, an analogous immunocytochemical labeling is present in the TN of the turbot, but whereas the specificity of the reaction in most of the turbot brain is confirmed by preabsorption experiments, in the TN the staining was not affected. This result suggests that the molecule recognized by the antiserum in this neural system is not NPY. Characterization of this peptide is beyond the scope of this study. Several lines of evidence indicate that FMRFamide-like immunoreactivity in the TN of fishes is due to the presence of F8Famide and A18Famide rather FMRFamide itself (Kyle et al., 1995; Fischer et al., 1996). If we take into account that the Cterminal amino acid sequence of these peptides differs
Table 1 Comparison of mean numbers of NPY like-ir neurons (including those in the olfactory organ, TN and olfactory bulbs) between either sides of the TN in metamorphic and juvenile turbots
Initial metamorphosis Intermediate metamorphosis Final metamorphosis Juvenile turbots
Number of NPY like-ir cells in the right side of turbot TN
Number of NPY like-ir cells in the left side of turbot TN
24 25 24 29
25 24 22 30
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Fig. 5. Brightfield photomicrographs showing the distribution of NPY like-ir cells in the terminal nerve of juvenile turbots. (A) A prominent group of NPY like-ir cells (arrowhead) can be seen in the left olfactory bulb that is on advance with respect to the right one. The asterisk indicates the olfactory nerve. Note that as consequence of the distortion, the plane of the section is horizontal throughout the olfactory bulbs, and sagittal in the rest of the brain. (B, C) Horizontal (B) and transverse (C) sections through the olfactory bulbs showing the NPY like-ir groups of cells (arrowheads). lO, left olfactory bulb; rO, right olfactory bulb; ON, optic nerve; T, telencephalon. Scale bars/230 mm in A, 100 mm in B, 50 mm in C.
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from that of NPY at only one position, it is likely that the antisera used in this study visualize these peptides and/or equivalent forms. Both spatial and temporal variations in the distribution of immunoreactive neurons in the TN of developing turbot are consistent with a pattern of migration of TN cells from the olfactory placode to the forebrain. Evidence that precursors of TN cells reside initially in the olfactory placode has been extensively demonstrated in tetrapods (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989a,b; Daikoku-Ishido et al., 1990; Ronnekleiv and Resko, 1990; Norgren and Lehman, 1991; Murakami et al., 1992; D’Aniello et al., 1994; Pellier and Astic, 1994). In fishes, early precursor cells of the TN have only been identified in the olfactory placode of teleosts (Parhar et al., 1995; Chiba et al., 1996a; Pinelli et al., 2000; present results), but the accurate origin of this system in other fishes remains unclear. Thus, whereas in cyclostomes the existence of the TN nerve is questioned (Bartheld et al., 1987; Eisthen and Northcutt, 1996), both in selaceans (Chiba et al., 1996a) and sturgeons (Pinelli et al., 2000), the rudimentary ganglion of the TN constitutes the first place in which cells expressing neuropeptides were detected. This indicates that in these species either the amount of the different neuropeptides in precursor cells of the olfactory placode was too low to be detected by immunocytochemical methods, or neuropeptide expression was delayed until cells migrate out of the olfactory organ. In turbot, first immunoreactive cells in the olfactory placode appear at hatching time, whereas in other teleost species TN cells containing different neuropeptides begin to differentiate during embryonic development (Parhar et al., 1995; Chiba et al., 1996a; Parhar and Iwata, 1996; Castro et al., 1999; Pinelli et al., 2000). The apparent delay observed in the emergence of TN cells in turbot must be correlated to the slow brain development in this fish, that also affects the emergence of other neural structures (Dolda´n et al., 2000). Soon after their generation, immunoreactive cells of the turbot TN begin to migrate to the brain, reaching an intracerebral location in the olfactory bulb. As occurs in most vertebrates the sequence of events involved in the development of the TN system in turbot occurs gradually, and it is not affected by the metamorphosis. Conversely, in salmon, the appearance of TN cells in the forebrain is delayed at the time of smolt-transformation (Parhar et al., 1995; Parhar and Iwata, 1996). In larval turbot, a significant development of the caudal projections of cells of the TN towards their targets is observed in temporal coincidence with the establishment of the cells inside the olfactory bulb, being apparently symmetrical and equal in both sides of the brain. However, the TN ontogeny is not completed during the larval period, since labeled neurons are observed in the olfactory organ until the metamorpho-
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sis. In fact, both cell migration and localization of TN cells in the olfactory bulb are strongly affected by the distortion suffered in the forebrain, although no clear differences in the number of immunoreactive cells were apparent in the TN at both sides of the brain. Additionally, TN axons proceeded to the telencephalic hemispheres by different routes. As a whole, variations of the TN components of the right and left side observed in this study, appear to be topological adaptations to the brain deformation. In the flatfish Pseudopleuronectes americanus , the olfactory system is strongly asymmetric (Prasada Rao and Finger, 1984). In this case, however, differences in the primary and secondary olfactory projections appear together with uneven extensions of the main target territories (the olfactory bulbs and the telencephalic hemispheres, respectively). That it is not the case for the TN, formed by a roughly similar contingent of neurons in both sides of the brain, that maintain connections with symmetric areas of the brain. Hence, it is conceivable that asymmetry in this neural system, if present, is significantly reduced.
Acknowledgements This work was supported by a grant from the University of Vigo UVIGO99 (64102C006). We thank Dr R. Anado´n, Dr J.M. Cerda´-Reverter and the anonymous reviewers for helpful comments on the manuscript, and we thank I. Emmett and A. Gonza´lez for the stylistic correction of the manuscript. Turbots were supplied by Piscı´cola del Morrazo (Nerga, Spain), Isidro de la Cal (Meira´s-Valdovin˜o, Spain), and the High Technology Center (Bergen, Norway).
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