Journal of Chemical Neuroanatomy 28 (2004) 37–46

Extrabulbar olfactory system and nervus terminalis FMRFamideimmunoreactive components inXenopus laevis ontogenesis

Claudia Pinellia, Biagio D’Aniellob,∗, Gianluca Poleseb, Rakesh K. Rastogib

a Department of Life Sciences, Second University of Naples, I-81100 Caserta, Italyb Department of Zoology, University of Naples Federico II, I-80134 Napoli, Italy

Received 20 November 2003; received in revised form 12 February 2004; accepted 1 June 2004

Abstract

The extrabulbar olfactory system (EBOS) is a collection of nerve fibers which originate from primary olfactory receptor-like neuronsand penetrate into the brain bypassing the olfactory bulbs. Our description is based upon the application of two neuronal tracers (biocytin,carbocyanine DiI) in the olfactory sac, at the cut end of the olfactory nerve and in the telencephalon of the developing clawed frog. Theextrabulbar olfactory system was observed already at stage 45, which is the first developmental stage compatible with our techniques; atthis stage, the extrabulbar olfactory system fibers terminated diffusely in the preoptic area. A little later in development, i.e. at stage 50, theextrabulbar olfactory system was maximally developed, extending as far caudally as the rhombencephalon. In the metamorphosing specimens,the extrabulbar olfactory system appeared reduced in extension; caudally, the fiber terminals did not extend beyond the diencephalon. While asubstantial overlapping of biocytin/FMRFamide immunoreactivity was observed along the olfactory pathways as well as in the telencephalon,FMRFamide immunoreactivity was never observed to be colocalized in the same cellular or fiber components visualized by tracer molecules.The question whether the extrabulbar olfactory system and the nervus terminalis (NT) are separate anatomical entities or represent an integratedsystem is discussed.© 2004 Elsevier B.V. All rights reserved.

Keywords: Ontogenesis; Brain; Amphibia; Biocytin; DiI; Olfactory system

1. Introduction

The extrabulbar olfactory projections (EBOP) are fibersthat originate from olfactory receptor-like neurons (ORN)and bypass the olfactory bulbs without terminating on themitral cells (Szabo et al., 1991). This fiber network is alsoknown as the extrabulbar olfactory system (EBOS) (Schoberet al., 1994). It should be emphasized that in many papers,previous to that ofSzabo et al. (1991), a system with EBOScharacteristics was described as the nervus terminalis (NT).A little later, however, some authors considered EBOS asa part of NT (Hofmann and Meyer, 1991b, 1995; Schoberet al., 1994; Meyer and Rastogi, 1998), whereas some otherssubstantiated the hypothesis that the two systems were dis-tinct neuroanatomical entities (Eisthen and Northcutt, 1996;Eisthen, 1997; Huesa et al., 2000; Laberge and Hara, 2001).

∗ Corresponding author. Tel.:+39 081 2535156;fax: +39 081 2535210.

E-mail address: biagio.daniello@unina.it (B. D’Aniello).

This dual interpretation is still vividly debated (seevonBartheld, 2004).

Except for embryonic (Santacana et al., 1992) and adultrats (Monti-Graziadei, 1992, 1993), as well as ducklings(von Bartheld et al., 1987; Meyer et al., 1987), a system withthe anatomical features of EBOS has only been reportedin totally or partially aquatic (fish and amphibian) species.Whereas the majority of these studies employed tracer sub-stances applied to several brain areas as well as in the ol-factory pit (Demski and Northcutt, 1983; Bazer et al., 1987;von Bartheld et al., 1986a,b, 1988; Schmidt et al., 1988;Hofmann and Meyer, 1989a,b, 1991a,b; Riddle and Oakley,1992), some investigators used immunohistochemical toolssuch as antibodies against various peptides (Honkanen andEkstrom, 1990, 1991; Szabo et al., 1987, 1991).

Quite a few species of amphibians have been investigatedin this regard (Schmidt et al., 1988; Hofmann and Meyer,1989a,b; Hofmann and Meyer, 1991a,b; Meyer et al., 1996;Koza and Wirsig-Wiechmann, 2001). From these reports itbecomes obvious that there are differences in the distribution

0891-0618/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.jchemneu.2004.06.001

38 C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46

of EBOS fibers in the sense that in some species this systemmay extend as far caudally as the mesencephalon (in someurodeles,Hofmann and Meyer, 1989a) whereas in many oth-ers such fibers show a shorter range of extension caudally,up to the diencephalon (Schmidt et al., 1988; Hofmann andMeyer, 1989b; Hofmann and Meyer, 1991a,b; Meyer et al.,1996; Koza and Wirsig-Wiechmann, 2001). Furthermore,some of these reports proposed that the EBOS is more devel-oped in species that rely on water born scent (seeHofmannand Meyer, 1989b). Nevertheless, this last hypothesis needsto be validated, given that the pathway was only recentlyrecognized, and has barely been discussed at all. In this con-text, an ontogenetic study could provide very useful infor-mation. Because of the paucity of similar studies we havecarried out a developmental study onXenopus laevis.

In the adultX. laevis, the NT with EBOS characteris-tics has been described to extend into the medial septum,preoptic nucleus, the nucleus of anterior commissure andthe hypothalamus (Hofmann and Meyer, 1989b). In thesame paper, the authors also examined a pool of tadpoleswhich the authors classified (based onNieukoop and Faber,1956) as stage 47; they applied an anterograde tracer(horseradish peroxidase, HRP) and very briefly stated thatthe NT in such tadpoles is “more developed” than in theadult.

The present study onX. laevis thus aims to provide amore detailed anatomical description of the EBOS duringontogenesis, and to determine whether or not the EBOS andthe FMRFamide-immunoreactive (ir) portion of the NT aredevelopmentally associated with each other. We have basedthis investigation on the application of tracers to visualizethe EBOS, and a widely recognized indicator of some com-ponents of the NT, the FMRFamide (molluscan cardioexci-tatory tetrapeptide).

The fluorescent carbocyanine dyes have been widely usedin cell biology to label neurons in vivo and in vitro and it hasbeen demonstrated that these lipophilic dyes are capable oftraveling along the cell membranes by diffusion (Honig andHume, 1986, 1989). Similar fluorescent lipophilic probeshave been successfully used aspostmortem neuronal trac-ers in aldehyde-fixed tissues (Godement et al., 1987; Honig,1993; Lukas et al., 1998) and they can be optimally appliedeither as anterograde or retrograde tracers (Kobbert et al.,2000). The EBOS revealed after anterograde tracer applica-tion in our samples extended posteriorly at least until thepreoptic area in all stages of development in which it wasinjected. Indeed, in order to retrogradely stain the ORNs ineach developmental stage studied, DiI was applied at thelevel of the anterior commissure. For DiI staining, however,the tissue is required to be placed in the fixative for a longperiod of time. Since immunohistochemical techniques usu-ally do not yield good results on over-fixed tissue, for doublestaining we used biocytin, and not DiI. Biocytin is appliedfor tracing in vivo and it is also used either as an antero-grade or retrograde tracer (Kobbert et al., 2000). It requiresa shorter incubation time and has a high affinity for avidin

that can be linked by fluorescent dyes coupled to avidin orstreptavidin.

Gonadotropin-releasing hormone (GnRH) and FMRF-amide-like ir elements have been described in the NTof several species of amphibians (Tuinhof et al., 1994;D’Aniello et al., 1994, 1996a,b; Iela et al., 1996; Pinelliet al., 1997; Rastogi et al., 1998, 2001; Fiorentino et al.,2001). Despite of the fact that they label only a part ofthe components of this nerve, they are considered the mostuseful NT markers (Wirsig-Wiechmann et al., 2002). SinceGnRH is not expressed duringX. laevis development priorto metamorphosis (Setalo, 1996), for the present study weselected FMRFamide-like immunolabel to identify one NTcomponent.

The results of this research may provide a wider basis fordiscussion and clarification of the definitions of the EBOSand/or the NT.

2. Materials and methods

2.1. Animals

Developmental stages (45, 47, 50, 51, 53, 54, 55, 56 and66) of the African clawed frog (X. laevis; Anura, Pipidae)were collected from a local breeding pool at the Univer-sity of Göttingen and classified according toNieukoop andFaber (1956). At least two samples of each stage were usedfor every variation of the technical procedures describedbelow.

2.2. Carbocyanine-dye DiI

After 4 h of fixation in 4% paraformaldehyde (PFA) in0.1 M phosphate buffered saline (PBS) pH 7.6, crystals ofDiI (1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanineperchlorate, Molecular Probes, OR, USA) were directlyapplied to the olfactory chamber or at the cut distal endof the olfactory nerves. In other samples, the brains weresectioned at the level of the anterior commissure/optic chi-asm, and the DiI crystals were applied to the entire exposedsurface of the transverse cut. Application sites were thencovered with a drop of agarose. In all cases the sampleswere maintained in PFA at 4◦C for 4–5 weeks.

After several washes in PBS the samples were embed-ded in 4% agarose and vibratome sectioned (Leica VT1000E). Horizontal and sagittal sections, 80�m-thick, were col-lected in PBS, mounted on glass slides and examined witha microscope.

2.3. Biocytin and FMRFamide immunohistochemistry:double staining

Under 0.03% tricaine methanesulfonate anesthesia (MS222, Sigma, St. Louis, USA) crystals of biocytin (Molecu-lar Probes) were applied into the nostrils with the help of a

C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46 39

thin needle, slightly damaging the olfactory epithelium tofacilitate the tracer incorporation. The Petri dish contain-ing biocytin-injected samples was kept on ice for 4 h. Thebrains were then removed and immersed in PFA for 4 hat 4◦C. For cryostat sectioning the samples were cryopro-tected in cold 30% sucrose solution in 0.1 M PBS overnight,placed in embedding medium (Tissue-Tek), frozen andstored at−20◦C until used. For vibratome sectioning, af-ter some washes in PBS, the samples were embedded in4% agarose.

In other samples the brains were decapsulated in frogsaline (Geiling and Schild, 1996) and the tracer was appliedto the ends of the olfactory nerve cut proximally to theolfactory mucosa as described byNezlin and Schild (2000).Briefly, a drop of paraffin oil was placed on the bottom of asilicon covered Petri dish, filled with saline and a small pieceof filter paper presoaked with saline was pinned inside. Thebrains were placed nearby and the cut ends of the nerveswere attached to the filter paper. Under a drop of paraffinoil, 3–4�l of 1% biocytin in dH2O were pipetted onto thefilter paper. After 1 h the brains were separated from thefilter paper and washed in fresh saline for 10–15 min. Thesamples were fixed and processed for cutting on a cryostator vibratome as described above.

Serial, sagittal or horizontal, cryostat sections (20�m)were mounted on polylysine-coated glass slides and storedat −20◦C before staining. The free-floating, sagittal andhorizontal vibratome sections (80�m) were collected inPBS. The sections stored at –20◦C were air-dried at roomtemperature for 45 min and rinsed in PBS (0.1 M, pH 7.6+ 3% Triton-X 100). Unspecific binding sites were blockedby 30 min incubation in PBS with 1% normal goat serumfollowed by incubation in 1% H2O2 in PBS for 30 minto block endogenous peroxidase activity. The sectionswere then rinsed in PBS and incubated with a solutionof avidin-Texas Red (1:150, Pierce, Rockford, IL, USA)or avidin Alexa-488 (1:200, Molecular Probes) in a darkmoist chamber (1–5 h) at room temperature. The same pro-cedure was used for the freefloating vibratome sections.After washing in PBS the sections were incubated withpolyclonal rabbit antibody against FMRFamide (1:1000,Diasorin, Stillwater, MN, USA) in a dark moist chamberovernight at 4◦C (72 h for thicker vibratome sections). Sub-sequently, the sections were rinsed several times in PBS andincubated for 1 h at room temperature (5 h for vibratomesections) either with fluorescein isothiocyanate-conjugatedgoat anti-rabbit antiserum (1:40, DAKO) when biocytinwas revealed by avidin Texas red, or with goat anti-rabbitTRITC (1:300, Pierce) when avidin Alexa was used in theprevious step. Finally, the sections were washed in PBSand mounted in glycerol/PBS (2:8) and examined in anepifluorescence or laser scanning confocal microscope.

Specificity tests were performed by omitting the primaryantiserum or by using a mixture of the antiserum previouslyincubated overnight with synthetic FMRFamide (5�M). Ineach case no staining was observed.

2.4. Microscopy and photodocumentation

The specimens were examined in an epifluorescent pho-tomicroscope (Axioscope, Zeiss, Germany) or a laser scan-ning microscope (LSM-510, Zeiss, Germany), equippedwith appropriate filter sets. For documentation, the Axio-scope was equipped with a photo camera. Black and whitenegatives (Ilford HP5 plus 400 ASA) were scanned at 1200dpi and exported as TIFF files. The digital images wereadjusted for brightness and contrast using Adobe Photo-shop 6. Plate photomontage and lettering were made usingCorelDraw 9.

3. Results

3.1. Anterograde tracing

No differences were observed in either the pattern of dis-tribution or the anteroposterior (cranio-caudal) extension ofthe fiber network when different tracers (DiI or biocytin)were applied among samples of the same stage of develop-ment. Nevertheless, in each case the staining was brightestwith DiI. The results obtained with the tracers put into theolfactory chamber are quite similar to those of biocytin ap-plication to the cut end of the olfactory nerve, and the follow-ing description is primarily based on the tracer applicationto the olfactory chamber. Any differences will be indicated.

When biocytin or DiI was applied to the olfactorychamber, the entire olfactory epithelium appeared heavilystained. Corresponding to the stain of the ORNs almost allfibers from the olfactory mucosa to the olfactory bulbs werestained. In addition, a neatly defined fiber network bypass-ing the olfactory bulbs was discerned caudally beyond theolfactory bulbs (Figs. 1A–C and 2A).

3.1.1. Stages 45, 47Some fiber bundles and several single fibers were ob-

served to bypass the olfactory bulbs, predominantly along amediobasal route. These fiber bundles were quite compactand thick (Fig. 2B) and continued ventromedially into thetelencephalon. They were observed to run as far caudal asthe anterior commissure where they spread and encircled thepreoptic recess. Some fibers ran across the anterior commis-sure to reach the contralateral side. No stained fibers wereseen further caudal to this area (Fig. 1A).

3.1.2. Stages 50–56A more extended fiber network was revealed after tracer

application to the olfactory mucosa. The fiber number shouldhave increased because the bundles appeared thicker as com-pared to the earlier stages. When biocytin was applied to thecut end of the olfactory nerve, some single labeled bipolarcell bodies in the telencephalic basal postbulbar area werealso noted (Fig. 3). As at previous stages, many fiber bundleswere located in the medioventral telencephalon and extended

40 C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46

Fig. 1. Schematic line drawings illustrating the extension of the EBOS asobtained by anterograde (A–C) and retrograde (D–F) DiI/biocytin tracingtechnique. (A and D) stage 45–47 (B and E) stage 50–56 (C and F) stage66. Abbreviations: AC: anterior commissure; APOA: anterior preopticarea; INF: infundibulum; MES: mesencephalon; OB: olfactory bulb; OM:olfactory mucosa; SM: medial septum.

as far caudally as the medial septum and the preoptic ar-eas (Fig. 2C). Most of the labeled fibers crossed at the levelof the anterior commissure. Some fiber endings were ob-served in the ventrolateral infundibulum as well (Fig. 2D).Many fibers, including some observed across the post-opticcommissure, ran caudally reaching the midbrain tegmen-tum. Only a few such fibers were observed to invade therhombencephalon (Fig. 1B).

3.1.3. Stage 66This stage, corresponding to the juvenile frog, showed

some differences with respect to the previous stages in asmuch as a general reduction in the distribution of the fiber

Fig. 2. (A–C) Imaged using a confocal microscope. (D) Imaged using aepifluorescent microscope. (A) Horizontal section of the brain of a stage54 sample showing EBOS central projections (arrowheads) after biocytinapplication to the olfactory mucosa. Rostral to the left. Asterisk shows theolfactory/vomeronasal nerve complex. OB: olfactory bulb. (B) Horizontalsection of the brain (rostral to the left) showing biocytin-stained fiberbundles and single fibers (arrowhead) in the mediobasal telencephalon,proximal to the anterior commissure (AC) after tracer application to theolfactory pit of a stage 47 sample. (C) Sagittal section of the brain ofa stage 51 sample showing EBOS fibers reaching the anterior preopticarea (arrowheads; inset is an enlargement). OB: olfactory bulb; TEL:telencephalon. (D) A horizontal section of the brain of a stage-53 tadpoleshowing some anterograde biocytin-stained fibers ending at the level of theventrolateral infundibulum (arrows), immediately behind the optic chiasm(asterisk). IR: infundibular recess. Rostral to the left. Scale bars= 50�m.

system was observed. The stained fibers terminated not farfrom the diencephalon, with a major concentration in thethalamus and hypothalamus and decreased everywhere innumber. On the other hand, the overall pattern of distributionat this stage closely resembles that of the developmentalstages 45 and 47 (Fig. 1C).

Fig. 3. Imaged using a confocal microscope. Biocytin-stained fibers anda cell body in the postbulbar basal telencephalon after tracer applicationto the cut ends of the olfactory nerve (stage 50). Rostral to the left. Scalebar = 10�m.

C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46 41

3.2. Retrograde tracing

When DiI was applied to the caudal telencephalon manystained fibers were seen to spread into the whole telen-cephalon. In all cases of retrograde staining we neverobserved the glomerular layer of the olfactory bulbs be-ing labeled. Rather, the fibers proceeded to the olfactorymucosa where several ORNs were stained (Fig. 4A and C).

3.2.1. Stages 45, 47Some stained fibers in the telencephalon crossed the ol-

factory bulbs, in their mediobasal part and, running alongthe medial part of the olfactory nerve, reached the olfactorymucosa, where a few ORNs were stained (Fig. 4A). They ap-peared elongated in shape without showing any preferentialdistribution in the olfactory epithelium. Another closely ly-ing group of cells, labeled after retrograde tracing, was exter-nal to the mediobasal part of the olfactory mucosa (Fig. 4B).This structure was only seen at the developmental stage 47(Fig. 1D).

3.2.2. Stages 50–56The neuroanatomical picture obtained was quite the same

as in stages 45 and 47. Nevertheless, proportional to the com-paratively higher number of stained fibers observed withinthe brain after anterograde tracing, a more conspicuous num-ber of olfactory fibers (Fig. 4D) and ORNs (Fig. 4C) were

Fig. 4. (A–D) Horizontal sections, imaged using a confocal microscope.(A) Retrograde stained olfactory receptor neurons (arrows) after DiI ap-plication at level of anterior commissure/optic chiasm section (Stage 47).Rostral to ward the top, and external to the left. OM: olfactory mu-cosa. (B) A group of cells retrogradely stained with DiI, proximal to themediobasal part of the olfactory mucosa (OM) (arrowhead; inset is anenlargement) of a 47 stage. Rostral to the right, and external toward thebottom. (C) Olfactory receptor neurons stained after DiI application tothe caudal telencephalon (stage 54). OM: olfactory mucosa. (D) Olfactoryfibers proximal to the olfactory mucosa stained after DiI application tothe caudal telencephalon (stage 54). Rostral toward the top, and externalto the leaft. Scale bars= 50�m.

labeled. In horizontal sections, the ORNs appeared moreconcentrated in the proximal part of the olfactory epithelium(Fig. 1E).

3.2.3. Stage 66The stained olfactory fibers crossing the olfactory bulbs

and the ORN numbers were less numerous as compared tothose in the previous stages of development (Fig. 1F).

3.3. Biocytin in the olfactory pit and FMRFamide-likeimmunohistochemistry double staining

The systems revealed by biocytin and by FMRFamideimmunohistochemistry appeared to overlap at the level ofthe olfactory/vomeronasal nerve complex, telencephalonand diencephalon. However, none of the fibers and cellbodies showed double-staining. Long FMRFamide-ir fibers(Fig. 5A) and cell bodies (Fig. 5B) could be distinguishedwithin the olfactory nerve, whereas biocytin labeled almostall the rest of the fiber components of the olfactory nerveand the related glomerular layers. In horizontal sections,immediately caudal to the olfactory bulbs, some medialectopic glomeruli intensely stained by biocytin could be dis-tinguished. FMRFamide-ir fibers ran across these glomeruli(Fig. 6A).

Caudal to the olfactory bulbs, many biocytin-labeledfibers, arranged in compact bundles, were observed en route

Fig. 5. (A–B) Imaged using an epifluorescent microscope. Long FMR-Famide immunoreactive fibers (A) and single cell bodies (B) within theolfactory nerve in a sample at stage 56. Rostral to the left. Scale bars30�m.

42 C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46

Fig. 6. (A–B) Imaged using a confocal microscope. Horizontal brain sec-tions showing the EBOS in red after biocytin injection into the olfac-tory camera and FMRFamide-ir fibers of the nervus terminalis in green,proximal to the olfactory bulb (A; rostral to the left) and at the ante-rior commissure level (B; rostral toward the top). Asterisk in A shows aglomerulus. Scale bars= 10�m in A, 20�m in B.

the mediobasal telencephalon. At this level, FMRFamide-irfibers were also present. They were predominantly locatedventral to the biocytin fibers. In horizontal sections, theFMRFamide-ir and biocytin-stained elements again sharedthe same anatomical regions (Fig. 6B). At the level of theanterior commissure, the biocytin-labeled compact ventro-medial bundles spread around the preoptic recess. Somefibers decussated to the contralateral side, while some othersran uncrossed posteriorly. At this level, the FMRFamide-irsystem was very rich in neurons and fibers.

The overlapping of biocytin and FMRFamide-like path-ways in the same brain areas, but without any colocalization,holds for each developmental stage studied. Regarding theFMRFamide-like ir system, we recorded an overall gradualincrease of stained elements in the brain during develop-

ment, whereas neurons and fibers decreased in the olfactorycomponents.

4. Discussion

Here, we report for the first time ontogenesis of the EBOSin an amphibian, the clawed frog (X. laevis) from an earlydevelopmental stage until after metamorphosis. The tracertechniques, together with FMRFamide immunohistochem-istry, revealed a substantial overlapping of label along theolfactory pathways as well as in the telencephalon. How-ever, FMRFamide immunoreactivity was never observed tobe colocalized in the same cellular or fiber components vi-sualized by tracer molecules.

4.1. Ontogenetic pattern

In this work onX. laevis we show that the EBOS could beobserved quite early during ontogenesis. Nevertheless, thetracer techniques employed in this research allowed us toonly visualize and study the EBOS starting from stage 45,since the earlier stages of development were not compatiblewith the surgical (tracer) technique. Therefore, the exacttime as to when during ontogenesis the EBOS elements firstappear remains undetermined. However, starting from thestage 45, the EBOS and the FMRFamide-ir components ofNT appeared as separate entities. The two systems did notappear to have any anatomical connection and this situationwas found unchanged throughout the larval development.

As far as the relative frequency of the tracer-labeled el-ements is concerned, EBOS developed progressively untilstages 50–56 and beyond, and it appears to undergo regres-sive changes plausibly during the metamorphic climax asascertained by the presence of a less developed system inthe juvenile frogs. This developmental pattern is consistentwith the observations ofHofmann and Meyer (1989b)fol-lowing HRP injection into the olfactory chamber ofX. lae-vis. These authors examined the adults as well as develop-mental stage-47 samples, and they simply mentioned thatthe tadpoles, as in our results, display a comparatively moredeveloped EBOS than in the adults. It is to be noted, how-ever, that the stage-47 pattern, as described byHofmann andMeyer (1989b), is quite similar to that observed in our tad-poles in developmental stages 50. This pattern shifting couldsimply be accounted for by a different stage classificationor by a different methodological approach, since stage 50differs from 47 only by the presence of a little undifferen-tiated limb bud (seeNieukoop and Faber, 1956). Indepen-dently from this controversy, it is evident that EBOS is moreextended during ontogenesis. This observation does not al-low us to propose any functional implication. It should bepointed out, however, that in cyclostomes, some primitivebony fishes and lungfishes, the EBOS fibers appear extendedmore caudally (von Bartheld and Meyer, 1988; Hofmann andMeyer, 1995), whereas in some more derived bony fishes

C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46 43

(euteleosts) such fibers do not extend beyond the forebrain(Demski and Northcutt, 1983; Bazer et al., 1987; Riddle andOakley, 1992; Becerra et al., 1994; Hofmann and Meyer,1995). These observations would lend support to the ideathat a reduction in the rostro-caudal extension of the EBOSfibers would occur already among water dwelling vertebratesand this concept may not be related to the transition of ver-tebrates from water to land (cf.Hofmann and Meyer, 1992).As such, we may presume that apparently there is an evo-lutionary trend for the EBOS to show a shorter extensionindependent of the habitat, water or land.

As early as 1911,Brookover and Jackson (1911)describedin a catfish that the NT develops from the olfactory placode.Particularly, a group of cells leaves the placode progressivelybecoming independent of it and ultimately forming the gan-glion of NT (GNT). The elaborate fiber system stems fromthe cells during their migration toward and into the brain.This mechanism was recently confirmed in several teleosts(Parhar et al., 1998; Castro et al., 1999, 2001; Pinelli et al.,2000; Prego et al., 2002; Amano et al., 2002; Pandolfi et al.,2002) as well as in the Australian lungfish (Fiorentino et al.,2002). Nevertheless, while the GNT proximal to the olfac-tory mucosa is present also in the adult lungfish (Schoberet al., 1994; Vallarino et al., 1995) and elasmobranchs (Locy,1905; White and Meredith, 1995; Moeller and Meredith,1998; Forlano et al., 2000), in teleosts it moves more cen-trally during development through the olfactory bulbs. Itmay form ganglia en route and often constitutes the nucleusolfactoretinalis in the brain. This nucleus was first describedby Munz et al. (1981)and is now known to be located in thepostbulbar basal telencephalon (Parhar et al., 1998; Pinelliet al., 2000; Prego et al., 2002; Amano et al., 2002; Pandolfiet al., 2002). In the present study as well, a group of cellsexternal but proximal to the olfactory mucosa was observed.Fiber projections originating from this cell group were visu-alized by the application of tracers into the brain, and as suchthis cell group could plausibly be considered a ganglion. Asimilar ganglion was only stained by the retrograde tracingtechnique in stage 47 tadpoles. This observation is partiallyconsistent with a previous study on the same species afterDiI injection into the diencephalon (Hofmann and Meyer,1992). Unfortunately, in this study the authors did not spec-ify at which stage of development they observed a similarfeature in the clawed frog.

The ganglion cells were never stained by FMRFamide im-munohistochemistry and therefore, we cannot be sure thatthis ganglion corresponds to the GNT of fish. This could alsobe due to the fact that the ganglion cells do not yet synthe-size sufficient amounts of FMRFamide-like molecules dur-ing this stage of development to be revealed by immunohis-tochemistry. At the same time, stained neurons, looking verysimilar to those usually observed in the NT, are found inthe olfactory nerve and forebrain after applying the tracersat the level of the cut olfactory nerve. It is well known thatFMRFamide-like immunoreactivity can be revealed at differ-ent stages during development in the olfactory components

of different species (seeFiorentino et al., 2001). For instance,in the zebrafish (Pinelli et al., 2000) FMRFamide-like im-munoreactivity in the NT was observed very early, whereasin the lizardChalcides chalcides it appears much later indevelopment (D’Aniello et al., 2001).

4.2. EBOS and NT: two systems?

Nervus terminalis was first described byFritsch (1878)asa supernumerary cranial nerve in a little shark. WhileLocy(1903)described ganglia amidst its fiber components, pres-ence of scattered neuronal cell bodies has been ascertainedin several species of vertebrates. In the majority of verte-brates, the NT appears as a loose fiber network originatingeither from ganglia or scattered single cells. In most cases theNT practically lies within the olfactory-vomeronasal nervescomplex (often anatomically indistinguishable) and conse-quently it is difficult to study its neuroanatomy. On the otherhand, in elasmobranchs and lungfishes, the NT is a compactbundle (with ganglia along its pathway) that runs separatelyfrom the olfactory nerve and thus it is easy to observe itsgross anatomy in a dissection or in traditional histologicalsections (Locy, 1903, 1905). For its immunohistochemicalproperties the NT can be visualized (at least partially) insome other vertebrates as well. Indeed, some substances (e.g.molecules belonging to FMRFamide, GnRH and NPY fam-ilies) have been observed to be selectively present in the NT,rather than in the olfactory or vomeronasal components ofthe olfactory-vomeronasal-terminal nerves complex. There-fore, they have been proposed as NT markers (seeDemski,1993; Wirsig-Wiechmann et al., 2002for review). How-ever, the immunohistochemical techniques pose two mainproblems: (1) such substances are also present in the fore-brain neurons which, with their fiber systems, make it diffi-cult to follow the central pathway of NT (seeMuske, 1993;Demski, 1993), and (2) all these markers are absent in theolfactory components in cyclostomes (Wicht and Northcutt,1992; Tobet et al., 1996; Eisthen and Northcutt, 1996).

These difficulties were supposed to be overcome in sev-eral studies addressed to demonstrate the presence of NTby application of tracers (e.g. HRP), carbocyanine dyesDiO, DiI or cobalt lysine) into the nasal sac of cyclostomes(Meyer et al., 1987; von Bartheld et al., 1987; Northcuttand Puzdrowski, 1988; von Bartheld and Meyer, 1988), am-phibians (von Bartheld and Meyer, 1986a,b; Schmidt et al.,1988; von Bartheld and Meyer, 1988; von Bartheld et al.,1988; Hofmann and Meyer, 1989a,b) and birds (Meyeret al., 1987). These data led some authors to conclude thatthe NT is a plesiomorphic feature of all vertebrate classes(von Bartheld et al., 1987; Hofmann and Meyer, 1989a,b;Fujita et al., 1991).

The discovery of an EBOS bySzabo et al. (1991)gener-ated remarkable difficulties of interpretation of all previousworks in which tracer substances applied to the olfactory sacwere used to demonstrate the presence of NT. Szabo et al.specifically indicated that a previous study in which HRP

44 C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46

was applied in the nasal sac ofPolypterus (von Bartheldand Meyer, 1986a) showed in reality the EBOS rather thanthe NT. They concluded that EBOS and NT are two distinctsystems and expressed serious doubts on the possibility ofidentifying the NT after applying tracers into the olfactorysac. Some authors tried to bypass this problem by consid-ering the EBOS as a subsystem of the NT (Hofmann andMeyer, 1991b, 1995; Schober et al., 1994; Meyer andRastogi, 1998). However, this view was strongly criticizedby others (Eisthen and Northcutt, 1996; Eisthen, 1997)who considered NT and EBOS as distinct neuroanatomicalentities.

In their review,Wirsig-Wiechmann et al. (2002)providean update on the definition of the NT on neurochemical, de-velopmental and neuroanatomical criteria where it is givento understand that the NT is a characteristic of gnathostomevertebrates. Basing their interpretation on the GnRH cells(Whitlock et al., 2003), the authors suggested that one ofthe major characteristics of the NT is that the neurons whichare integral part of the nerve, arise from the neural crestcells that move very early into the olfactory placode andmigrate from there into nasal and ventral forebrain areas.Regarding the neurochemical aspects, at least a part of NTcomponents should contain a form of GnRH, acetylcholineand/or NPY/FMRFamide-like compound. Anatomically in-stead, the NT would be characterized by the presence of cellbodies that lie within the nasal cavity and rostroventral ar-eas of the brain whose axons project anteriorly towards theolfactory organ and centrally to the brain. Particularly, thelast definition would leave little space to the considerationthat EBOS could be a part of NT as previously postulated(Hofmann and Meyer, 1991b, 1995; Schober et al., 1994;Meyer and Rastogi, 1998).

The present study onX. laevis provides some evidence tosupport the view that EBOS and the FMRFamide-ir com-ponent of the NT, at least in the amphibians, are separateanatomical entities. Simultaneous use of neuronal tracersand FMRFamide immunohistochemistry has shown a sub-stantial overlapping of the two systems. It is important tonote, however, that we never observed a colocalization ofthe tracer and FMRFamide-like material.

In amphibians, it was earlier demonstrated that the NTlocated FMRFamide-ir fibers bypassing the olfactory mu-cosa and contacting the olfactory chamber lumen are absentin the adult green frog (Rana esculenta: D’Aniello et al.,1996a) and the common toad (Bufo bufo: Fiorentino et al.,2001). Transitory NT FMRFamide ir fibers amidst the recep-tors cells of the olfactory epithelium were described in earlystages ofR. esculenta tadpoles (D’Aniello et al., 1996a).From these ontogenetic data in green frog and in the com-mon toad, as well as the fact that FMRFamide ir cells havenever been reported in the olfactory epithelium of any am-phibian species during the adulthood (seeFiorentino et al.,2001), it is reasonable to assume that the FMRFamide NTsystem does not originate anatomically from cells of the ol-factory epithelium. The position of FMRFamide fibers ex-

ternal to the olfactory epithelium, probably renders it moredifficult for them to incorporate tracer molecules put intothe olfactory sac. This view is also shared byHuesa et al.(2000) who applied some tracer substances in the chon-drosteanAcipenser baeri to study the EBOS.

Two distinct anatomical pattern were also revealed by us-ing GnRH immunohistochemistry as the NT marker and bio-cytin injection into the olfactory chamber ofX. laevis (Kozaand Wirsig-Wiechmann, 2001), or GnRH/HRP in the samespecies (Hofmann and Meyer, 1991b) and in the Africanlungfish,Protopterus dolloi (von Bartheld et al., 1988). Fur-ther support for this view derives from studies in whichtracer substances were used in those species in which theNT is clearly separated from the olfactory nerve, and thusvisible without the use of any particular staining: elasmo-branchs (Hofmann and Meyer, 1995) and a lungfish (vonBartheld et al., 1988). In these cases, besides the staining ofthe EBOS, the tracers put in the olfactory sac were not in-corporated in any NT ganglia or cell bodies. Thus, it seemsthat FMRFamide and GnRH do not stain neuronal cell bod-ies amidst the fiber tracts stained with neuronal tracers.

However, in the adultX. laevis some neurons in the ol-factory nerve labeled after biocytin application in the ol-factory sac were considered as part of the NT (Koza andWirsig-Wiechmann, 2001). The authors could discern suchcells in moderately labeled olfactory nerve bundles. It wasnot possible to verify this observation in our preparationsbecause of a constant heavy staining of the olfactory nerve.

In some elasmobranches (Demski et al., 1987), as wellas in the teleosts,Clarias batrachus (Subhedar and RamaKrishna, 1988) and Cirrhinus mrigala (Biju et al., 2003),GnRH was found to stain at least some part of the olfactoryepithelium. Occasional GnRH-ir cell bodies were describedin the vomeronasal and olfactory epithelia ofAmbystomatigrinum (Wirsig-Wiechmann, 1993). Biju et al. (2003)hy-pothesised that the GnRH-ir fibers observed in the forebrainof C. mrigala might belong to EBOS. If this were true, andif we accept GnRH as an NT marker, we should considerEBOS as a part of NT. Unfortunately, the authors did notuse any tracer substances, but there is good reason to be-lieve that GnRH-ir olfactory receptor cells could incorporatetracers placed in the olfactory chamber.

It is obvious that the presence of GnRH-ir elements inthe olfactory mucosa renders it more difficult to generalize adistinction between EBOS and NT. Thus, further studies arerequired in those species in which GnRH stains the ORNs,combining immunohistochemistry and tracer techniques, toclarify whether the GnRH-ir cells have characteristics ofEBOS cells or if they have to be considered as non-migratedelements of NT.

Acknowledgements

We thank Prof. C. von Bartheld for editing and criti-cal reading of the manuscript and Dr. L.P. Nezlin for his

C. Pinelli et al. / Journal of Chemical Neuroanatomy 28 (2004) 37–46 45

technical expertise, particularly with LSM and for his criti-cal comments. Contributions from Programma Vigoni, Italy(R.K.R.) and DDA, Germany (D.L. Meyer) are acknowl-edged. C.P. acknowledges the postdoctoral fellowship fromthe Alexander von Humboldt Foundation, Germany. The au-thors wish to express their gratitude to late Prof. D.L. Meyerand dedicate this study to him.

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