Centrifugal visual system ofCrocodylus niloticus: A hodological, histochemical, and...

Centrifugal Visual System of Crocodylusniloticus: A Hodological, Histochemical,

and Immunocytochemical Study

MONIQUE MEDINA,1* JACQUES REPERANT,2,3 ROGER WARD,3,4

AND DOM MICELI3,4

1Centre National de la Recherche Scientifique UMR8570-MNHN USM0302,F-75005 Paris, France

2Hopital de la Salpetriere, Institut National de la Sante et de la Recherche Medicale U106,F-75013 Paris, France

3Centre National de la Recherche Scientifique UMR5166-MNHN USM0501,F-75005 Paris, France

4Universite du Quebec a Trois Rivieres, Groupe de Recherche en Neurosciences, TroisRivieres, Quebec G9A 5H7, Canada

ABSTRACTThe retinopetal neurons of Crocodylus niloticus were visualized by retrograde transport of

rhodamine beta-isothiocyanate or Fast Blue administered by intraocular injection. Approxi-mately 6,000 in number, these neurons are distributed in seven regions extending from themesencephalic tegmentum to the rostral rhombencephalon, approximately 70% being locatedcontralaterally to the injected eye. None of the centrifugal neurons projects to both retinae. Theretinopetal neurons are located in rostrocaudal sequence in seven regions: the formatio reticu-laris lateralis mesencephali, the substantia nigra, the griseum centralis tectalis, the nucleussubcoeruleus dorsalis, the nucleus isthmi parvocellularis, the locus coeruleus, and the commis-sura nervi trochlearis. The greatest number of cells (approximately 93%) is found in the nucleussubcoeruleus dorsalis. The majority are multipolar or bipolar in shape and resemble the ectopiccentrifugal visual neurons of birds, although a small number of monopolar neurons resemblingthose of the avian isthmo-optic nucleus may also be observed. A few retinopetal neurons in thegriseum centralis tectalis were tyrosine hydroxylase (TH) immunoreactive. Moreover, in thenuclei subcoeruleus dorsalis and isthmi parvocellularis, both ipsilaterally and contralaterally,approximately one retinopetal neuron in three (35%) was immunoreactive to nitric oxide syn-thase (NOS), and a slightly higher proportion (38%) of retinopetal neurons were immunoreactivefor choline acetyltransferase (ChAT). Some of them contained colocalized ChAT and NOS/reduced nicotinamide adenine dinucleotide phosphate–diaphorase. Fibers immunoreactive toTH, serotonin (5-HT), neuropeptide Y (NPY), or Phe-Met-Arg-Phe-amide (FMRF-amide) werefrequently observed to make intimate contact with rhodamine-labeled retinopetal neurons. Thesefindings are discussed in relation to previous results obtained in other reptilian species and inbirds. J. Comp. Neurol. 468:65–85, 2004. © 2003 Wiley-Liss, Inc.

Indexing terms: crocodilian; retinopetal neurons; ChAT; NOS/NADPHd; TH

A centrifugal visual system (CVS), consisting of projec-tions from the brain to the retina, was described in birdsat the end of the nineteenth century (Ramon y Cajal, 1889;Dogiel, 1895; Wallenberg, 1898). The existence of similarretinopetal projections in nonavian species was the objectof extensive controversy until the development of experi-mental tracing and immunohistochemical methods almosta century later, and a CVS has been described in membersof all vertebrate groups, including man (Reperant andGallego, 1976; Reperant et al., 1989; Uchiyama, 1989;Ward et al., 1991; Rio, 1996). More specifically, in reptiles,this system has been demonstrated in turtles (Schnyder

Grant sponsor: Centre National de la Recherche Scientifique; Grantsponsor: Institut National de la Sante et de la Recherche Medicale; Grantsponsor: Fonds de Recherche Sur la Nature et les Technologies; Grantsponsor: Conseil de Recherches en Sciences Natureiles et en Genie duCanada.

*Correspondence to: Monique Medina, Laboratoire d’Anatomie Compa-ree, Museum National d’Histoire Naturelle, 55 rue Buffon, 75005 Paris,France. E-mail: [email protected]

Received 19 March 2003; Revised 1 August 2003; Accepted 19 August2003

DOI 10.1002/cne.10959Published online the week of November 10, 2003 in Wiley InterScience

(www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 468:65–85 (2004)

© 2003 WILEY-LISS, INC.

and Kunzle, 1983; Weiler, 1985; Schutte and Weiler, 1988;Malz and Meyer, 1994; Reiner et al., 1996), lizards (Halp-ern et al., 1976; Kenigfest et al., 1986; Malz and Meyer,1994; El Hassni et al., 1997), snakes (Halpern et al., 1976;Reperant et al., 1980a, b, 1981; Hoogland and Welker,1981; Schroeder, 1981), and crocodiles (Ferguson et al.,1978). In the latter group, data are available only forCaiman crocodilus. Ferguson et al. (1978) describe in thisspecies, the presence of neurons retrogradely labeled bi-laterally by horseradish peroxidase (HRP) in the isthmicregion.

The first objective of the present investigation, carriedout in a second species, Crocodylus niloticus, was to obtainadditional information concerning the crocodilian CVS. Inaddition to a quantitative study of retrogradely labeledneurons after intraocular injections of rhodamine beta-isothiocyanate (RITC) or Fast Blue (FB), we present theresults of several double-labeling and triple-labeling ex-periments intended to show, by immunofluorescence,which neuroactive substances or their enzymes are in-volved in the CVS of C. niloticus, by testing for the pres-ence of several substances that have already been identi-fied in the CVS of other vertebrate species (cf. Reperant etal., 1989; Uchiyama, 1989; Ward et al., 1991; Miceli et al.,1999). The second objective was to undertake a compara-tive analysis of our results, together with those of theliterature, to define the status of the CVS of Crocodyluswith regard to that of other reptiles and that of birds, agroup generally considered to be phylogenetically close tocrocodiles (Walker, 1972; Martin, 1983).

MATERIALS AND METHODS

Eight specimens of Crocodylus niloticus, between 10and 12 months of age, weighing 200–300 g, were obtainedfrom the “Ferme aux Crocodiles”, Pierrelatte, France. Theanimals were maintained in filtered aquaria in the labo-ratory. All experimental procedures were carried out inaccordance with the guidelines of the European and Ca-nadian Councils of Animal Care.

Retrograde labeling

Under ketamine hydrochloride/Rompun anesthesia (50,20 mg/kg, respectively), each animal received a unilateralintraocular injection of 40 �l of a 25% (w/v) solution ofRITC (Sigma–Aldrich) containing 2% dimethyl sulfoxide.

In three animals, 40 �l of a 5% solution of FB (Sigma–Aldrich) was injected into the other eye. Injections weremade with a 50-�l Hamilton syringe whose needle was leftin place for 3 min before its careful removal.

After a survival period of 10–18 days, at a constanttemperature of 30°C, the animals received an overdose (1ml) of Nembutal and were then perfused transcardiallywith 0.9% heparinized saline followed by 4% paraformal-dehyde in 0.1 M phosphate buffer (PB; pH 7.4) at a flowrate of 10 ml/min. Brains were removed and stored over-night in fresh fixative at 4°C, then in 10% sucrose in PBfor 6 hours and finally in 30% sucrose in PB overnight.Coronal sections were cut at 25 �m in a Leitz-Lauda 1720cryostat. Sections were gathered onto uncoated slides, de-hydrated, cover-slipped, and examined with an Aristoplanmicroscope using N2 (580 nm) and H3 (520 nm) filter-mirror system to detect rhodamine and FB fluorescence,respectively.

Topographical procedures

Coronal sections 250 �m apart, from the most rostral tothe most caudal planes in which labeled cells were ob-served, were photographed on Ilford HP5 film by using a10� objective. Prints were made at various enlargements,and each section was reconstructed by photomontage. Toshow the cytoarchitecture of regions in which labeled cellswere observed, sections were then rehydrated, stainedwith cresyl violet, recover-slipped, and photographed un-der brightfield illumination on Tmax 400 Pro film, andeach section was reconstructed once more by photomon-tage. Drawings indicating the boundaries of the differentcytoarchitectural features were made by using Adobe Pho-toshop and Adobe Illustrator software packages.

Cell counts

These were carried out in three specimens, in which thepopulations of centrifugal neurons labeled with Fast Bluewere estimated. It is to be noted that cells labeled withrhodamine present a bright homogeneous red fluorescencethroughout the cell body, whereas Fast Blue–labeled cellsdisplay diffuse light blue and silver intense granular flu-orescence throughout the cytoplasm; this labeling beingvery weak in the nucleus, which is thus clearly discernible(Bentivoglio et al., 1980; Illert et al., 1982; present inves-tigation). The total number of labeled cells, contralateraland ipsilateral, were estimated in serial sections 25 �m

Abbreviations

Aq aqueductCb cerebellumCoNIV commissura nervi trochleariscRN contralateral retinopetal neuronsGCT griseum centralis tectalisFLM fascicularis longitudinalis medialisFRL formatio reticularis lateralis mesencephaliIco nucleus intercollicularisIP nucleus interpeduncularisiRN ipsilateral retinopetal neuronsIS nucleus isthmiISm nucleus isthmi magnocellularisISp nucleus isthmi parvocellularisLC locus coeruleusLL nucleus lemniscus lateralisNIII nucleus nervi oculomotoriiNIV nucleus nervi trochlearis

NVmd nucleus motorinus nervi trigemini pars dorsalisNVmv nucleus motorinus nervi trigemini pars ventralisNR nucleus raphesOS nucleus olivaris superiorOT optic tectumPrm nucleus profundus mesencephaliSCd nucleus subcoeruleus dorsalisSCda nucleus subcoeruleus dorsalis anteriorSCdm nucleus subcoeruleus dorsalis medialisSCdp nucleus subcoeruleus dorsalis posteriorSCdpa nucleus subcoeruleus dorsalis posterior, pars anteriorSCdpc nucleus subcoeruleus dorsalis posterior, pars caudaleSCv nucleus subcoeruleus ventralisSN substantia nigraTS torus semicircularisV ventricleVTA ventral tegmental area

66 M. MEDINA ET AL.

thick from the most rostral (tegmentum) to the most cau-dal (isthmus) region containing retinopetal neurons. Ob-servations were made under epifluorescence (H3 filter)using 25� and 40� objectives. Counts were made both byeye and in digitized images taken by using a Sony cameraand Optilab software, inspired by the method of opticaldissection (West, 1993; Coggeshall and Lekan, 1996).Thus, only those Fast Blue–labeled cells containing nu-clear profiles within the upper optical plane of the sectionwere recorded; those containing nuclear profiles withinthe lower optical plane of the section were not. Individualcell nuclei observed within both optical planes of any givensection were also included in the count. The counts wereperformed throughout the section where retrogradely la-beled and immunostained neurons were located and werecarried out by sampling sections at constant intervals inthe rostrocaudal plane of between every 5 and 10 sections,depending on the brain structure. Unbiased estimates ofthe total population of labeled neurons, and more pre-cisely of their relative distributions (percentage of total)bilaterally within different structures of the brainstem,were obtained by multiplying the values of neuron num-ber recorded within each structure in the sampled sectionsby the sampling interval number (West, 1999).

Histochemical procedures

Those sections containing retrogradely labeled neuronswere further treated for reduced nicotinamide adenine

dinucleotide phosphate–diaphorase (NADPH-d) activityaccording to the procedure described by Morgan andMiethke (1994). Sections were detached from their slidesand then incubated in Tris buffer (0.05 M, pH 7.6) con-taining 0.2% Triton X-100, 0.2 mM nitroblue tetrazolium(NBT, Sigma), and 1 mM beta-NADPH (Sigma), either atroom temperature (27°C) or at 37°C, for 30 minutes. Sec-tions were then rinsed in Tris buffer, mounted onSuperfrost-plus slides, air-dried for 30 minutes, then re-hydrated for 60 seconds and cover-slipped with Vectash-ield. They were examined under brightfield conditions todetect the NADPH-d reaction product.

Immunocytochemical procedures

Those sections in which retrogradely labeled neuronswere visible were detached from their slides and incu-bated for 48 hours at 4°C in one of the following primaryantibodies: anti-serotonin (5-HT) 1:150 or 1:2500, anti–neuropeptide Y (NPY) 1:1000, anti–tyrosine hydroxylase(TH) 1:250 or 1:500, anti–�-aminobutyric acid (GABA)1:3,000 or 1:10,000, anti–nitric oxide synthase (NOS) -I1:250 or 1:800, anti–choline acetyltransferase (ChAT) 1:100,anti–Phe-Met-Arg-Phe-amide (FMRF-amide) 1:2,000, oranti–gonadotropin-releasing hormone (GnRH) 1:1,000 (seeTable 1). They were subsequently incubated in a fluorescein-conjugated secondary antibody for 2 hours at room temper-ature, either Silenius goat anti-rabbit immunoglobulin IgG1:200, Chemicon rabbit anti-goat IgG 1:50, or Molecular

TABLE 1. Antisera Used in Immunocytochemical Investigations1

Antiserum HostSource1 and

code Dilution Immunogen Specificity analyzed by References

Serotonin (5-HT) Rabbit ImmunotechCode 0601

1:150 Serotonin formaldehyde BSA Recognizes serotonin informaldehyde-fixed tissuesections

Steinbusch et al., 1978

Serotonin (5-HT) Rabbit Sigma–AldrichCode S5545

1:2500 Serotonin–creatinine sulfate complexconjugated with formaldehyde toBSA

Reacts with serotonin-containing fibers inparaformaldehydeperfusion-fixed, frozensections

Smeets and Steinbusch, 1988

Neuropeptide Y (porcine)(NPY)

Rabbit PeninsulaCode IHC7172

(T-4454)

1:1000 Synthetic NPY (poroine) Den Boer-Visser andDubbeldam, 2002

Medina et al., 1992Tyrosine hydroxylase (TH) Rabbit Chemicon

Code AB1521:250 Denatured TH from rat

pheochromocytoma (denatured bysodium dodecyl sulfate)

Western blot Brauth, 1988

Tyrosine hydroxylase (TH) Rabbit Jacques BoyCode

208020234

1:500 Pure TH from rat pheochromocytoma Test of Ouchterlony andWestern blot

Arluison et al., 1984

Nitric oxide synthase-I(bNOS)

Rabbit ChemiconCode AB1552

1:250 Synthetic peptide corresponding toamino acid residues 1414–1429 ofrat NOS-I protein conjugated toKLH via a maleimido linkage

Western blot of ratcerebellum and cortexshows a strong band 155kDa

Rachman et al., 1996

Nitric oxide synthase-I(bNOS)

Rabbit CederlaneCode B220-1

1:800 Synthetic peptide from C-terminal ofcloned rat cerebellar NO-synthasecoupled to BSA

Absorption with 10 100 �gimmunogen per ml dilutedantiserum abolishes thestaining

Alm et al., 1993Miceli et al., 1999

�-Aminobutyric acid (GABA) Rabbit Sigma-AldrichCode A2052

1:30001:10000

GABA coupled to BSA Reacts with GABA andGABA-KLH. dot blotassay. No reaction withBSA

Pow et al., 1995

Choline acetyltransferase(ChAT)

Goat ChemiconCode AB144P

1:100 Human placental enzyme ImmunoblotWestern blot

Anadon et al., 2000; Brauthet al., 1985

FMRF-amide (Phe-Met-Arg-Phe.amide)

Rabbit AffinitiCode FA1155

1:2000 Synthetic FMRF-amide coupled toBSA using glutaraldehyde to yieldan N-terminal bound conjugate

Blocking immunostaining bypreadsorbtion with 10nmol synthetic FMRFamide per ml dilutedantibody

D’Aniello et al., 1999

Gonadotropin-releasinghormone (GnRH) cloneSMI41

Mouse AffinitiCode GA1184

1:1000 GA1184 antibody (mouse IgG) reactswith GnRH producing cells inseveral mammalian species

Reacts with GnRH at anaffinity of 10�11 M andspecific for C-terminalpenta-peptide (Gly-Leu-Arg-Pro-Gly.NH2)

Oka and Ichikawa, 1990

1Sources include Chemicon International, Temecula, CA; Sigma–Aldrich, Inc., Saint Louis, Missouri; Immunotech, Marseille, France; Cederlane, Ontario, Canada; Affiniti, Exeter,UK; Peninsula, Belmont, California; Institute Jacques Boy s.a., Reims, France. BSA, bovine serum albumin.

67RETINOPETAL NEURONS OF CROCODYLUS

Probes Alexa Fluor 488 goat anti-rabbit IgG/donkey anti-goat IgG 1:100, rinsed in PBS, gathered onto slides andcover-slipped with Vectashield or Vectashild-DAPI. Sectionswere examined under ultraviolet light by using the followingbarrier filters: A (360 nm) to detect fast blue, N2 (580 nm) todetect rhodamine, and I3 (488 nm) to detect fluorescein.They were also examined under a confocal microscope (seebelow). Control sections were subjected to the same proce-dure but with the omission of the primary antibody. Underthese circumstances, a complete absence of immunolabelingwas observed in all structures examined.

Triple-labeling procedures

Those sections with RITC-labeled neurons were firsttreated with the ChAT antibody (Chemicon), according tothe same protocol listed above and visualized by using theFITC-labeled secondary antibody (Chemicon). Then thesesections were rinsed in Tris-buffer and incubated in asolution of 0.2 mM NBT and 1 mM beta-NADPH with0.2% Triton X. The NADPH reaction times were varied toobtain a purple color that could easily be distinguishedfrom the labeling produced by the two fluorochromes.These three colors obtained by immunohistochemicalstaining and histochemistry allowed us to distinguishthree labels in the same section.

Confocal microscopy

A Nikon Eclipse 600 inverted confocal laser scanningmicroscope (CLSM) was used, in conjunction with theBio-Rad MRC-1024 scanning system and a krypton–argonlaser emitting at 488 nm to excite fluorescein and 568 nmto excite rhodamine. Observations were made with a Ni-kon planapochromat 20� objective with a numerical ap-erture of 0.50, and confocal images were collected bymeans of LaserSharp software running under the OS/2operating system.

Terminology

The nomenclature used in this report is, with minormodifications, that proposed by Brauth (1988) and Derob-ert et al. (1999).

RESULTS

Distribution of retinopetal neurons

In all specimens injected with rhodamine, the antero-grade transport of this tracer heavily labeled all the brainareas known to receive primary visual projections (Derob-ert et al., 1999), with no indication of any trans-synaptictransport of RITC. In addition, clearly retrogradely la-beled neurons were observed, bilaterally but with con-tralateral predominance (68%), throughout a region ap-proximately 4–5 mm long, extending from themesencephalic tegmentum to the rostral rhombencepha-lon. The retinopetal neurons (RN) are located, in rostro-caudal sequence, in seven distinct regions: (1) the formatioreticularis lateralis mesencephali (FRL), (2) the griseumcentralis tectalis (GCT), (3) the substantia nigra (SN), (4)the nucleus subcoeruleus dorsalis (SCd), (5) the nucleusisthmi parvocellularis (ISp), (6) the locus coeruleus (LC),and (7) the commissura nervi trochlearis (CoNIV; Fig.1A–P).

Cell counts in three specimens revealed a mean numberof 4,219 (� 880 � SD) contralaterally labeled RN (cRN)

and of 1,953 � 441 ipsilaterally labeled RN (iRN) giving atotal estimate of 6,172 � 707 retinopetal neurons. Thesmallest number of RN (cRN � iRN) was observed in theFRL (0.4%) and CoNIV (0.4 %), and in increasing order inthe SN (0.6%), LC (0.7%), GCT (1.5%), ISp (3.5%), and SCd(92.9%; Table 2). This latter structure can be subdividedinto three components (Fig. 1A–L): the most rostral(SCda) at the level of the substantia nigra and NIII, thecentral SCdm at the level of the central and posteriorregions of NIII, and the most caudal SCdp at the level ofNIV. The SCdp can in turn be subdivided into two regions:one caudal (SCdpc), facing the ISp, and one more rostral(SCdpa), where the ISp is absent. Among these, the great-est number of RN was observed in the SCdp (70.2%),progressively fewer being observed in the SCdm (16%) andSCda (6.7%). The distributions of contralaterally (cRN)and ipsilaterally (iRN) labeled neurons in these structuresappear to be comparable (Table 2).

Morphology of retinopetal neurons

In those specimens having received an injection of RITCin one eye and FB in the other, no double-labeled neuronscontaining both tracers were observed. Each RN thusprojects to one eye, without collateral branches passing tothe other (Fig. 2).

The cell bodies of these neurons were either multipolaror bipolar, giving rise to smooth processes that branchedsparsely, producing relatively simple dendritic arboriza-tion. The somata of multipolar neurons were ovoidal, tri-angular, or polygonal, from which arose three to five pri-mary dendritic trunks (Fig. 3D–F). These cells areextremely variable in size, with minor diameters and ma-jor diameters ranging, respectively, from 18 �m to 30 �mand 22 �m to 45 �m. Bipolar RN were generally fusiformin shape, with minor and major diameters ranging from10 �m to 12 �m and 16 �m to 21 �m. From each extremityof the soma arose a single, long dendritic extension withfew ramifications (Fig. 3C,D). These two cell types wereobserved intermingled with each other in essentiallyequal numbers, with the larger cell bodies being less fre-quently observed. The third category of RN, considerablyfewer in number than the other two, had pyriform or ovoidmonopolar somata, bearing a tuft of two or three den-drites; from the opposite pole of the soma, the beginning ofan axon could sometimes be observed (Fig. 3A,B,D); theaxon could, occasionally, be seen to arise from one of theprimary dendritic trunks. These neurons, with somaticminor and major diameters ranging, respectively, from 15�m to 30 �m and 20 �m to 38 �m, were most often seen inthe caudal SCdpc.

Immunocytochemistry

Our initial observations sought to determine which neu-roactive substances could be demonstrated in the cell bod-ies and fibers of those structures in which RN were ob-served. In none of these were neurons immunoreactive toFMRF-amide or to GnRH observed. Cell bodies immu-nopositive to NPY were observed in the SN; ChAT-positivesomata in the SCd, ISp, LC, and CoNIV; and 5-HT–positive cell bodies in the LC and the ventral part of theSCdp. Neurons immunoreactive to TH were present in theSN, GCT, SCd, and ISp. NADPH-d�/NOS-immunoreactive (-ir) and GABA-ir cells were observed ineach of the seven structures containing retinopetal neu-rons (Table 3; Figs. 4, 5). Large numbers of axons immu-

68 M. MEDINA ET AL.

noreactive to 5-HT, TH, NPY, ChAT, NOS, or FMRF-amide were also observed in these structures, particularlyin the SCdp (Table 4; Figs. 4, 5).

Multiple labeling

Of the antibodies we used, only those directed againstNOS, ChAT, and TH gave immunolabeling of RITC-labeled retinopetal neurons on both sides of the brain(Tables 1, 5). Double labeling with anti-NOS or NADPH-dhistochemistry and RITC was observed in all regions (Ta-ble 5; Figs. 4, 6, 7A). Double labeling of RITC-labeledretinopetal neurons with anti-ChAT was observed only inSCd, ISp, LC, and CoNIV (Table 5; Figs. 4, 7B). In thoseregions containing the greatest numbers of contralaterallyand ipsilaterally labeled retinopetal neurons, the SCd(containing approximately 93% of the total number of RN)and the ISp (containing approximately 3.5% of the totalnumber of RN), we estimated the proportion of centrifugalvisual neurons labeled by either of these antibodies. Ineach of the subdivisions of the SCd (SCda, SCdm, SCdp)and in the ISp, approximately one retinopetal neuron inthree (35% of the population of cRN and iRN in each ofthese structures) was immunoreactive to NOS (Table 2)and a slightly higher proportion (38%) of cRN and iRN wasChAT-immunoreactive (Table 2). In triple labeling, someof these cell bodies also showed NADPH-d immunoreac-tivity (Figs. 4, 8).

In the GCT, a few rhodamine-labeled retinopetal neu-rons were TH-ir (Fig. 9). All of the antibodies used, withthe exception of those directed against GABA and GnRH,produced immunolabeling of fibers, particularly withinthe SCdp (Table 4; Figs. 4, 5).

By combining corresponding RITC and FITC imagesobtained with the confocal microscope, it was possible toinvestigate the distributions of TH-ir, 5-HT-ir , NPY-ir,and FMRF-amide-ir fibers which traversed the SCdp inthe region of retrogradely labeled retinopetal neurons.The immunolabeled fibers were frequently observed to beapposed to the somata or dendritic processes; of these,there were no unlabeled pixels between red and greenstructures in the appositions, which could be observed inseveral consecutive planes (Fig. 7C,D). This finding sug-gests that these appositions were not accidental, and re-flected the close contact between fibers immunoreactive tothese substances and the centrifugal visual neurons.

DISCUSSION

Relations to previous findings in Caiman

After an intraocular injection of HRP, Ferguson et al.(1978) observed, in Caiman crocodilus, retrogradely la-beled cells essentially in the isthmic region. These cellswere distributed bilaterally but with a contralateral pre-dominance. They formed a simple, large, oblique cell field,occupying two regions: one, described as Group Y by Hu-ber and Crosby (1926), in which the numerous centrifugalneurons were tightly packed, and a second, more exten-sive internal region bounded medially by the nucleustrochlearis, within which the density of labeled neuronswas lower. These regions correspond, respectively, to theISp and the SCdp of Crocodylus niloticus. Nevertheless, inthe latter species, in contrast to Caiman, the density oflabeled neurons is higher in the internal region than inthe external one. In neuromeric nomenclature (Medina et

Fig. 1. Schematic drawings of transverse sections of the brain of asingle specimen of Crocodylus niloticus, 250 �m apart, passing cau-dally from the mesencephalon (A) to the isthmic region (P), showingthe distribution of retinopetal neurons (RN). Each RN is representedby a single large dot. Retinal terminals in the superficial tectal layersare represented by small dots. For abbreviations, see list. Scale bar �0.5 mm in C,G,L,P (applies to A–P).


al., 1993; Puelles, 1995, 2001), the two regions arise fromthe isthmus (alar plate) or rhombomere 0.

Our use of RITC as a tracer revealed a much greaterrostrocaudal extent of the population of centrifugal neu-rons, extending caudally from the tegmentum mesen-cephali arising from the mesomere (FRL, SN, GCT,SCda, SCdm) to the posterior isthmus (LC, CoNIV).Three possible explanations for the differences betweenthe two species may be offered: that they reflect inter-specific differences, that they arise from the use of twodifferent tracers, or that the more extensive distribu-tion of labeled cells in Crocodylus is the result of trans-synaptic transport of RITC. It has, for example, beenshown in the pigeon (Miceli et al., 1993, 1997), thatafter intraocular injection of RITC with long (more than21 days) survival times, the tracer can leak out of thecentrifugal neurons to be taken up by the terminals oftheir afferent supply and transported retrogradely totheir cell bodies, particularly in the tectum. We can

eliminate the third possible explanation for two rea-sons: on the one hand, in our material the number ofretrogradely labeled neurons did not vary with survivaltimes between 10 and 18 days, and on the other hand,Ferguson et al. (1978) have shown that the centrifugalvisual neurons of Caiman receive their afferent supplyfrom the tectum, and we imagine that the same is trueof Crocodylus. We point out that we observed no cellularlabeling in the tectum after the longest survival time of18 days; hence, we conclude that the labeling of neuronsoutside the isthmic region is not the result of trans-synaptic transport but indeed the result of direct retro-grade transport from the retina. The labeled cells, thus,are retinopetal centrifugal neurons.

In the optic nerves of Alligator missippiensis andCaiman crocodylus, Kruger and Maxwell (1969) ob-served, 20 months after enucleation, approximately4,000 myelinated fibers with relatively normal appear-ance, representing approximately 5% of the total popu-

Figure 1 (Continued)

70 M. MEDINA ET AL.

lation of myelinated axons. Given the slow rates oforthograde and retrograde degeneration in reptiles(Armstrong, 1951; Kruger and Maxwell, 1969; Reperantand Rio, 1976; Reperant et al., 1991), their conclusionthat these fibers were retinopetal appears entirely jus-tified and is supported by the data of Ferguson et al.(1978) and the present results. We note that the number

of supposedly centrifugal fibers described by Kruger andMaxwell (1969) corresponds fairly well to the number ofretrogradely labeled neurons in Crocodylus.

Ferguson et al. (1978) briefly describe two types of cen-trifugal neurons in Caiman, multipolar and bipolar fusi-form cells. We have observed these two types of cell inCrocodylus, providing additional information as to their


72 M. MEDINA ET AL.

sizes, and we also describe a third, less common, monopo-lar type of retinopetal neuron.

In Caiman, as in Crocodylus, the retrogradely labeledneurons are found bilaterally, and it may be imagined thatthey project to both ipsilateral and contralateral retinae.Although Ferguson et al. (1978) provide no data relevantto this question, we have shown, by the use of two differ-

ent tracers (FB and RITC) that, in Crocodylus, no centrif-ugal neuron bears collateral fibers innervating the tworetinae.

Relations to previous findings in otherreptilian species

Location of centrifugal neurons.

Chelonians. In all species of turtle that have beenexamined (Schnyder and Kunzle, 1983; Weiler, 1985;Schutte and Weiler, 1988; Malz and Meyer, 1994; Reineret al., 1996; Haverkamp and Eldred, 1998), between 10and 80 centrifugal neurons have been identified, situatedbetween the mesencephalic tegmentum and the anteriorrhombencephalon. The most detailed description is pro-vided by Haverkamp and Eldred (1998), who used the betasubunit of cholera toxin as a tracer in Trachemys scriptaelegans. Of the 40 neurons they counted, approximately90% were situated contralaterally in the tegmentum, inthe FRL, SN, and in a region ventral to the latter possiblycorresponding to the SCda and SCdm of crocodiles, thesestructures arising from the mesomere. They also observed,in the isthmic region arising from rhombomere 0, severalcentrifugal neurons in the ISp, in and around the locuscoeruleus, in the nucleus reticularis isthmi and the supe-rior raphe. The nucleus reticularis isthmi, at least itsanterior part, may well be equivalent to the SCdp of croc-odiles.

While the total number of centrifugal visual neurons inturtles is considerably smaller, by a factor of 100, thanthat in crocodiles, the distribution of these cells is compa-rable in the two groups, with the exception that retinope-tal neurons are absent from the superior raphe of thelatter.

Lacertilians. In all species that have been examined(Halpern et al., 1976; Kenigfest et al., 1986; Malz andMeyer, 1994; El Hassni et al., 1997), a population of cen-trifugal visual neurons, whose number varies betweenspecies, has been observed in the isthmic region arisingfrom rhombomere 0. A second population of retinopetalneurons has been described in some species (Halpern etal., 1976; El Hassni et al., 1997) in the ventral thalamusarising from prosomere 3. The distribution of centrifugalneurons in lizards, thus, is somewhat different from thatin crocodiles.

Ophidians. In Henophidian snakes, centrifugal vi-sual neurons have been observed in the basal telenceph-alon and the preoptic area arising from prosomere 6(Hoogland and Welker, 1981). In contrast, in all Co-

Fig. 2. Light microscopy. Darkfield photomicrographs taken ofretrogradely labeled neurons in the right SCdm of a specimen ofCrocodylus niloticus that had received an intraocular injection ofrhodamine beta-isothiocyanate (RITC) into the left eye (thin arrows)and a similar injection of Fast Blue (FB) into the right eye (thickarrows). Note that no labeled neuron shows both fluorescent tracersand, hence, that none of these cells project to both retinae. Scalebars � 100 �m in A,B.

TABLE 2. Neurons Located within the Brainstem1

StructuresNumberof RN

Percentageof RN

ncRN

Percentages of contralateral RN

niRN

Percentages of ipsilateral RN

cRN

cRNChAT-positive

cRNNOS-

positive iRN

iRNChAT-positive

iRNNOS-

positive

FRL 28 0.4 19 0.3 - 0.1 9 0.1 - -SN 38 0.6 27 0.4 - 0.1 11 0.2 - -GCT 93 1.5 64 1.0 - 0.3 29 0.5 - 0.1SCda 420 6.7 293 4.7 1.7 1.6 127 2.0 0.7 0.7SCdm 965 16.0 727 12.0 4.5 4.2 238 4.0 1.5 1.4SCdp 4332 70.2 2882 46.7 17.7 16.3 1450 23.5 8.9 8.2ISp 220 3.5 145 2.3 0.8 0.8 75 1.2 0.4 0.4LC 48 0.7 22 0.6 - 0.2 6 0.1 - -CoNIV 28 0.4 40 0.3 - 0.1 8 0.1 - -

1Percentages of the total number of retinopetal neurons (RN) and of ChAT-positive and NOS-positive neurons located contralaterally (cRN) and ipsilaterally (iRN) within differentstructures of the brainstem of Crocodylus niloticus. ChAT, choline acetyltransferase; NOS, nitric oxide synthase. For other abbreviations, see list.


enophidian species that have been examined (Halpernet al., 1976; Reperant et al., 1980a, b, 1981; Schroeder,1981), the retinopetal neurons are located bilaterally inthe ventral thalamus (prosomere 3), as they are in somelizards. In Vipera aspis (Reperant et al., 1980a, b, 1981,1989), approximately 660 such neurons have been de-scribed, representing approximately 1% of the numberof fibers in the optic nerve (Ward et al., 1989). Thedistribution of centrifugal visual neurons in snakes,thus, differs widely from that seen in crocodiles.

Neurochemical aspects. The antibodies against neu-roactive substances or their enzymes of synthesis used inthis study have been used previously to identify the cellbodies or fibers of the centrifugal visual system in othervertebrate species: GnRH in teleosts (Munz et al., 1982;Stell et al., 1984, 1987; Oka and Ichikawa, 1990; Oka andMatsushima, 1993); FMRF-amide in teleosts (Stell et al.,1984, 1987) and anuran amphibians (Wirsig-Wiechmanand Basinger, 1988; Uchiyama et al., 1988); NPY in te-leosts (Vecino and Ekstrom, 1992; Chiba, 1997); TH in

Fig. 3. Light microscopy. Darkfield photomicrographs showing the different types of retinopetalneurons of Crocodylus niloticus. Monopolar (A,B,D), bipolar (C,D) and multipolar (D–F) neurons areindicated respectively by small, medium, and large arrows. Scale bar � 100 �m in F (applies to A–F).

74 M. MEDINA ET AL.

anuran amphibians (Schutte and Witkowsky, 1991) andmammals (Simon et al., 2000); 5-HT in chondrichthyans(Ritchie and Leonard, 1983; Schlemermyer and Chappell,1991), teleosts (Lima and Urbana, 1998), anurans(Schutte and Witkowsky, 1990), turtles (Schutte andWeiler, 1988), and mammals (Villar et al., 1987; Schutte,1995; Reperant et al., 2000); GABA in petromyzontiforms(Rio et al., 1992, 1993, 1996; Vesselkin et al., 1996); NOS/NADPH-d in turtles (Blute et al., 1997; Haverkamp andEldred, 1998) and birds (Miceli et al., 1999); glutamate inpetromyzontiforms (Rio et al., 2002b) and birds (Rio,1996); and ChAT in birds (Bagnoli et al., 1992; Medinaand Reiner, 1994; Miceli et al., 1999). The immunocyto-chemical and histochemical data of the present study in-dicate that, of all these substances, only NOS/NADPH-d,ChAT, and TH are located in the centrifugal visual neu-rons identified by retrograde labeling with RITC. We havealso shown that NADPH-d and ChAT may be colocalizedwithin the same neuron; thus, we conclude that NO andacetylcholine are used either singly or jointly as neuro-transmitters by some of the centrifugal visual neurons ofcrocodiles.

Analyses of immunoreactivity to several of the antibod-ies used in this study have been carried out in severalreptilian brain species: (1) 5-HT in snakes (Challet et al.,1991), lizards (Wolters et al., 1985; Smeets and Stein-busch, 1988; Bennis et al., 1990a; Pierre et al., 1990), andturtles (Ueda et al., 1983); (2) TH or dopamine in Caiman(Brauth, 1988), snakes (Smeets, 1988b), lizards (Smeets etal., 1986; Smeets, 1988a, 1994; Bennis et al., 1990b; Lopezet al., 1992; Medina et al., 1994), turtles (Smeets et al.,1987); (3) ChAT in turtles (Powers and Reiner, 1993) andlizards (Medina et al., 1993); (4) NPY in lizards (Medina etal., 1992; Bennis et al., 1999); (5) NOS/NADPH-d in liz-ards (Smeets et al., 1997); and (6) FMRF-amide in turtles(D’Aniello et al., 1999), lizards (Vallarino et al., 1994), andCaiman (D’Aniello et al., 1999). Compared with our ownimmunocytochemical data, the studies of other reptilianspecies show essentially the same distribution of immu-noreactivity of cell bodies and fibers to these substances inthose regions equivalent to those of the crocodile in whichcentrifugal visual neurons are found.

Few immunocytochemical or histochemical studies havesought to identify the neurochemical properties of reptil-ian centrifugal visual neurons, with the exception of thosecarried out in the turtle Trachemys scripta elegans(Weiler, 1985; Schutte and Weiler, 1988; Yaqub and El-dred, 1991; Blute et al., 1997; Haverkamp and Eldred,

1998). Weiler (1985) reports enkephalin immunoreactivityin approximately one third of the centrifugal visual neu-rons identified by nuclear yellow labeling in the isthmicregion, and Schutte and Weiler (1988) found a singleserotonin-containing efferent neuron in the same region ofthe brain. The latter type of centrifugal visual neuron wasnot found in our investigation. Furthermore, Yaqub andEldred (1991) observed 4 to 10 aspartate-containing effer-ent nerve fibers in the retina of Trachemys but did notdescribe the origin of these fibers. In addition, Blute et al.(1997) describe 7 to 10 NADPH-d/NOS–reactive efferentfibers in the retina of this species. In both cases,aspartate-immunoreactive and NOS-immunoreactive fi-bers arborize extensively over several millimeters of ret-ina. Finally, Haverkamp and Eldred (1998) have shownthat, as in the crocodile, approximately 30% of the retino-petal neurons of Trachemys are NADPH-d–positive. Theseauthors also point out that the distribution of NADPH-d–positive efferent cells overlaps that of ChAT-ir neurons(Medina and Reiner, 1994), and they suggest that theymay also use acetylcholine as a neurotransmitter. Thishypothesis is confirmed by the present results. In contrastto Crocodylus niloticus, no TH-ir retinopetal neurons havebeen identified in other reptilian species, although theymay exist in amphibians (Schutte and Witkovsky, 1991)and in mammals (Simon et al., 2000).

Comparison with birds

It is generally accepted that a close evolutionary linkexists between crocodiles and birds (Walker, 1972; Martin,1983). A comparative analysis of the centrifugal visualsystems of the two, thus, is obviously called for.

Morphologic aspects. The well-developed retinopetalsystem of birds comprises two populations of neurons; thewell-defined nucleus isthmo-opticus (NIO), and a popula-tion of ectopic retinopetal cells (EC). These two formationsare located in the isthmic region arising from rhombomere0. The NIO is dorsally located, bounded medially by thetrochlear nucleus and laterally by the isthmic nuclei par-vocellularis (ISp) and magnocellularis (ISm); the ectopiccentrifugal neurons are scattered diffusely around theNIO, mainly ventral to it in the tegmental reticular field.The number of neurons in the NIO varies somewhat be-tween species; 8,000 to 12,000 in ground-feeding birds(Cowan, 1970; Reperant et al., 1989), 3,000 to 6,000 inbirds with a well-developed trigeminal system (Sohal,1976; Reperant et al., 1989), and 900 to 2,000 in raptorsand birds that feed on the wing (Shortess and Klose, 1975;

TABLE 3. Detection of Substances within Cells1

Structures

Antisera

TH 5-HT NOS NADPH-d GABA ChAT NPY FMRF-amide GnRH

FRL � � �� SN �� GCT �� SCda �� SCdm �� SCdp �� ISp � �� LC �� CoNIV � � ��

1Immunocytochemical detection of eight neuroactive substances or their enzymes of synthesis within cells of brainstem structures containing retinopetal neurons retrogradelylabeled with RITC or FB. ��, very high density; ��, high density; ��, medium density; �, low density. RITC, rhodamine beta-isothiocyanate; FB, Fast Blue; TH, tyrosinehydroxylase; 5-HT, serotonin; NOS, nitric oxide synthase; NADPH-d, reduced nicotinamide adenine dinucleotide phosphate–diaphorase; GABA, �-aminobutyric acid; ChAT,choline acetyltransferase; NPY, neuropeptide Y; FMRF-amide; Phe-Met-Arg-Phe-amide; GnRH, gonadotropin-releasing hormone. For other abbreviations, see list.


Fig. 4. Schematic drawings of a transverse section through theisthmic region (SCdpc) of Crocodylus niloticus, situated between sec-tions K and L of Figure 1, showing the distribution of retinopetalneurons (unfilled circles) and the distribution of cholineacetyltransferase–immunoreactive (ChAT-ir) cell bodies (solid black

circles) and fibers of passage (above) and of nitric oxide synthase orreduced nicotinamide adenine dinucleotide phosphate–diaphorase(NOS/NADPH-d) -reactive cell bodies (below) and fibers of passage.The retinopetal somata that colocalize the two enzymes of synthesisare indicated by stars. For abbreviations, see list. Scale bar � 0.5 mm.

76 M. MEDINA ET AL.

Weidner et al., 1987; Reperant et al., 1989; Feyerbende etal., 1994). In granivores, the NIO appears as a highlyconvoluted laminar structure formed of two layers of cen-trifugal visual neurons separated by a layer of neuropil

(Cowan, 1970; Miceli et al., 1995). The majority of theneurons are monopolar with flask-shaped or pyriform so-mata, 15–25 �m in diameter, from the apical pole of whichtwo to four primary dendritic trunks emerge to divide into

Fig. 5. Schematic drawings of the same section as in Figure 4,showing the distribution of contralateral retinopetal neurons (unfilledcircles) and the distribution of fibers of passage (solid black) immu-noreactive to different neuroactive substances or to their enzymes ofsynthesis. Neurons immunoreactive to serotonin (5-HT), tyrosine hy-droxylase (TH), or �-aminobutyric acid (GABA) are present in theSCdpc but are not retinopetal (left-hand column). No neurons immu-

nopositive to Phe-Met-Arg-Phe-amide (FMRF-amide), neuropeptide Y(NPY), or gonadotropin-releasing hormone (GnRH) are present in theSCdpc, but this region is traversed by fibers immunoreactive to thesesubstances. Terminal arborizations reactive to FMRF-amide or NPYmay contact the centrifugal neurons. A similar distribution of theseelements was observed ipsilaterally. For other abbreviations, see list.Scale bar � 1 mm.


parallel branches directed toward the neuropil. Their ax-ons arise either from the opposite basal pole of the cellbody or from one of the apical dendritic trunks (Cowan,1970; Miceli et al., 1995; Li and Wang, 1999a). The NIOalso contains a small population of GABAergic interneu-rons (Miceli et al., 1995). In adult birds, the centrifugalneurons of the NIO project essentially to the contralateralretina. Within the retina, the majority of centrifugal axonsend in restricted, or convergent, terminals which form apericellular nest around associative amacrine cells(Ramon y Cajal, 1893; Maturana and Frenk, 1965; Hayesand Webster, 1981; Woodson et al., 1995). The NIO is anessential component of a retino-tecto-isthmo-retinal feed-back loop (Cowan, 1970).

The ectopic centrifugal neurons are fewer in numberthan those of the NIO, the ratio EC/NIO varying from 15%to 30%, depending on the species (Hayes and Webster,1981; O’Leary and Cowan, 1982; Weidner et al., 1987,1989; Reperant et al., 1989). They form a heterogeneouspopulation of multipolar and fusiform bipolar cell bodies,ranging from 10 �m to 35 �m in equivalent diameter. Inadult birds, their projections to the retina are predomi-nantly contralateral, with a minority of ipsilaterally pro-jection neurons whose proportion varies from 2% to 27%,depending on the species (Hayes and Webster, 1981;O’Leary and Cowan, 1982; Weidner et al., 1987, 1989).Double-labeling studies (Weidner et al., 1987, 1989; Rep-erant et al., 1989) have shown that, in adult birds, none ofthese cells possesses collateral branches projecting to thetwo retinae. The axons of the ectopic neurons form, in theretina, divergent or widespread terminals, which arehighly collateralized, extending over wide areas of theinferior retina, making contact for the most part withdisplaced ganglion cells (Nickla et al., 1994).

In Crocodylus niloticus, no convoluted structure resem-bling the avian NIO is formed by the retrogradely labeled

centrifugal visual neurons. On the other hand, the wide-spread dispersion of these neurons and the multipolar orfusiform shape of the majority of their somata closelyresemble the features of the ectopic centrifugal neurons ofbirds. In addition, while the centrifugal neurons of theavian NIO project exclusively to the contralateral retina,the avian ectopic neurons project to both ipsilateral andcontralateral retina, as do the retinopetal neurons of croc-odiles; however, the proportion of ipsilaterally projectingcentrifugal neurons in crocodiles (32%) is somewhathigher than that in birds (2–27%). In both groups, thecentrifugal visual neurons are not collateralised and donot project to both retinae.

On the other hand, in the crocodilian SCdp, there existsa small population of “tufted” monopolar neurons, whosemorphology closely resembles that of the monopolar neu-rons of the avian NIO. However, these cells do not projectexclusively to the contralateral retina and are regroupedin a recognizable structure but appear to be scattered atrandom within the SCdp. It may be the case, therefore,that, in the crocodilian, SCdp lies the precursor of theavian NIO. It should be borne in mind that, among thebirds, the NIO of the raptors is poorly differentiated, fre-quently reticular, and contains fewer neurons than that ofother avian groups (Shortess and Klose, 1975; Weidner etal., 1987; Reperant et al., 1989). This type of organizationof the NIO, thus, may represent an intermediate stagebetween that seen in crocodiles and in granivorous birds.

The distribution of centrifugal visual neurons in birds(NIO, EC) is limited to the isthmic region, derived fromrhombomere 0, whereas in crocodiles, these neurons areobserved not only in this region (SCdp, ISp, LC, CoNIV),but also in more rostral regions derived from the mesom-ere (FRL, SN, GCT, SCda, SCdm). On the grounds of aterminology based on neuromeric topography, we, there-fore, advance the hypothesis that the crocodilian SCdp is

TABLE 5. Colocalization of Immunoreactivities1

Structures

Antisera

TH 5HT NOS NADPH-d GABA ChAT NPY FMRF-amide GnRH

FRL � � � � � � � �SN � � � � � � � �GCT � � � � � � � �SCda � � �� SCdm � � �� SCdp � � �� ISp � � � � � � � �LC � � � � � � � �CoNIV � � � � � � � �

1Colocalization of NOS/NADPH-d, ChAT, and TH immunoreactivities within retinopetal neurons labeled with RITC or FB. For abbreviations, see Table 3 and the list.

TABLE 4. Detection of Substances within Fibers1

Structures

Antisera

TH 5HT NOS NADPH-d GABA ChAT NPY FMRF-amide GnRH

FRL � � � � � �� SN �� GCT �� SCda �� SCdm �� SCdp �� ISp � �� LC �� CoNIV ��

1Immunocytochemical detection of eight neuroactive substances or their enzymes of synthesis within fibers of brainstem structures containing retinopetal neurons. Forabbreviations, see Table 3 and the list.

78 M. MEDINA ET AL.

the homologue of the avian NIO and EC. In both cases, theretinopetal neurons are situated in the isthmic region,bounded laterally by NIV, and, more caudally, medially bythe isthmic nuclei, and in both cases, the cells are the keystructure in a retino-tecto-isthmo-retinal feedback loop(Cowan, 1970; Ferguson et al., 1978; Reperant et al., 1989;Miceli et al., 1997, 1999). Furthermore, the crocodilianSCdp is the only structure in this species in which mo-nopolar neurons resembling those of the avian NIO are tobe found, and the number of centrifugal neurons observedin this structure (approximately 4,000 retinopetal neu-rons) is somewhat greater than the number found in ra-pacious birds (Shortess and Klose, 1975; Weidner et al.,1987; Reperant et al., 1989).

No data are available concerning the mode of terminalarborization of centrifugal visual fibers in the retina of thecrocodile. We speculate that it may be found that themajority of these fibers make divergent arborization, as dothe axons of the avian ectopic neurons, while the axons ofthe tufted monopolar neurons make convergent arboriza-tion, as do those of the neurons of the avian NIO.

Immunocytochemical and histochemical aspects.

As in crocodiles, no centrifugal visual neurons of birds(NIO, EC) have been described as 5-HT-ir (Cozzi et al.,1991; Challet et al., 1996; Medina et al., 1998), NPY-ir(Medina et al., 1998), nor as GABA-ir (Miceli et al., 1995).However, in marked contrast to birds, in which they areabsent (Medina et al., 1998), a few retinopetal neurons ofCrocodylus are TH-ir. On the other hand, several studiesin birds have shown that, as in the crocodile, some cen-trifugal visual neurons are ChAT-immunopositive, NOS-immunopositive, or NADPH-d–positive. Medina and

Reiner (1994) indicate that, in the pigeon, the size andlocation of ChAT-ir neurons around the NIO correspond tothose of the ectopic centrifugal neurons; in the same spe-cies, Bagnoli et al. (1992) and Miceli et al. (1999) provideevidence for a restricted population of ChAT-positive cellswithin the NIO, both suggesting that the ectopic retino-petal neurons are also ChAT-positive. In contrast, noChAT immunoreactivity has been found in the centrifugalvisual neurons of the chicken (Sorenson et al., 1989) orquail (Medina et al., 1998). Studies of nitric oxide in theavian brain, either by NOS immunocytochemistry orNADPH-d histochemistry, have demonstrated its pres-ence in the centrifugal visual neurons (Brunning, 1993;Meyer et al., 1994; Morgan et al., 1994; Montagnese andCsillag, 1996; Cozzi et al., 1997; Fischer and Stell, 1999;Miceli et al., 1999), although NADPH-d–positive cellshave not been reported in the NIO of the quail (Medina etal., 1998). As in the crocodile, the high degree of overlap ofthe distributions of ChAT-positive and NOS/NADPH-d–positive neurons suggests that both neuroactive sub-stances may be colocalized within the same cells (Miceli etal., 1999). As in Crocodylus (Medina et al., 1998; Miceli etal., 1999; Rio et al., 2002a), the centrifugal visual neuronsof birds appear to receive afferent supplies from fibersimmunoreactive to ChAT, NOS, 5-HT, TH, and NPY (Me-dina et al., 1998).

In conclusion, Crocodylus niloticus shares, with birds(Miceli et al., 1999) and turtles (Haverkamp and Eldred,1998), centrifugal visual neurons that are ChAT-ir andNOS-ir/NADPH-d–positive. Retinopetal neurons usingthese neuroactive substances have not been reported in

Fig. 6. Light microscopy. Photomicrographs of a section throughthe SCdp of Crocodylus niloticus (darkfield in A, brightfield in B),showing the distribution of contralateral retinopetal neurons labeledwith rhodamine beta-isothiocyanate (RITC, A) and the distribution ofreduced nicotinamide adenine dinucleotide phosphate–diaphorase

(NADPH-d) -reactive cell bodies (B).One labeled retinopetal neuroncontains the NADPH-d reaction product (large arrow) and twoNADPH-d–immunoreactive neurons are not retinopetal (small ar-rows). For abbreviation, see list. Scale bar � 100 �m in A (applies toA,B).


other vertebrate groups and, therefore, may be a charac-teristic of the Sauropsidae.

The widespread distribution of centrifugal visual neu-rons in crocodiles, extending from derivatives of the me-somere to derivatives of rhombomere 0, resembles thatobserved in turtles. On the other hand, the retinopetalneurons of birds are restricted to derivatives of rhom-bomere 0. Nevertheless, the centrifugal visual system ofcrocodiles bears some similarities to that of birds; the

number of retinopetal neurons (900–12,000 in birds, ap-proximately 6,000 in crocodiles) is considerably higherthan in other vertebrates, and an important proportion ofthese are involved in a retino-tecto-isthmo-retinal feed-back loop. In both crocodiles (present results) and birds(Challet et al., 1991; Medina et al., 1998), in contrast toturtles (Schutte and Weiler, 1988), no centrifugal visualneurons are 5-HT–immunopositive. In addition, in bothbirds and crocodiles, some centrifugal visual neurons have

Fig. 7. Confocal laser scanning images of the SCdp of Crocodylusniloticus showing double-labeled rhodamine beta-isothiocyanate(RITC) /nitric oxide synthase (NOS) –immunoreactive (-ir; arrows inA) and RITC/choline acetyltransferase (ChAT) -ir (arrows in B) con-tralateral retinopetal neurons. Numerous cell bodies (green) are ei-ther NOS-ir or ChAT-ir. C,D: The retrogradely labeled retinopetal

neurons (red) are surrounded by fibers of passage (green), some ofwhich give off small buttons (yellowish red) appearing to make con-tact with the somata and dendrites of the retinopetal neurons (ar-rows). For abbreviation, see list. Scale bars � 50 �m in A,B; 20 �m inC,D.

80 M. MEDINA ET AL.

a particular morphology, that of the monopolar tuftedretinopetal neurons, which have never been described inother reptilian groups. The plan of organization of thecrocodilian CVS, thus, may be considered as intermediatebetween that of chelonians and that of birds.

Functional considerations

We have shown that approximately 35–38% of the cen-trifugal visual neurons of Crocodylus are NOS-ir/NADPHd-positive or ChAT-ir, and that a proportion ofthese cells contain both of these enzymes of synthesis.

Nitric oxide (NO) seems to play an important role as aneurotransmitter or neuromodulator and intracellularmessenger in the central and autonomic nervous systemsof vertebrates (Bredt and Snyder, 1992; Snyder and Bredt,1992; Vincent and Hope, 1992). It may act either as a

neurotransmitter after its presynaptic release (Gartwaite,1991), or as a retrograde messenger modulating presyn-aptic activity after its postsynaptic release (reviews inGartwaite, 1991; Bredt and Snyder, 1992; Wilklund et al.,1993). That NO synthesizing enzymes are found in a cer-tain proportion of the retinopetal neurons of Crocodylussuggests that it plays a non-negligible role in the centrif-ugal visual system of these species. Among its possiblefunctions, it is possible that the NO released by centrifu-gal terminals in the retina activates guanyl cyclase in theretinal ganglion cells and modifies the conductivity of thecGMP channels that have been demonstrated in thesecells (Ahmad et al., 1994). It is also possible that thereleased NO may modulate gap junctions between ama-crine cells (Mills and Massey, 1995).

The possible role of the ChAT-positive neurons is harderto explain. Acetylcholine may have either excitatory orinhibitory effects, depending on the nature of the postsyn-aptic target (review in Ravel et al., 1990), although it isgenerally believed to play an excitatory role in the visualsystem (Pasik et al., 1990, for review), and it may wellexert the same function in crocodilian retinopetal neu-rons.

Fig. 8. Light microscopy. Photomicrographs of a section throughthe SCdp of Crocodylus niloticus illustrating an example of triple-labeling. A: Darkfield image of contralateral retinopetal neurons la-beled with rhodamine beta-isothiocyanate (RITC) are observed.B: Brightfield image of reduced nicotinamide adenine dinucleotidephosphate–diaphorase (NADPH-d) -reactive neurons. C: Brightfieldimage of choline acetyltransferase (ChAT) -immunopositive neuronsare observed. A single retinopetal neuron (large arrow) colocalizesChAT and NADPH-d; another is ChAT-reactive (small arrow), Tworetinopetal neurons (arrowheads) are neither NADPH-d–reactive norChAT-positive; three neurons (stars) are neither ChAT-reactive norretinopetal. Scale bar � 50 �m in C (applies to A–C).

Fig. 9. Light microscopy. Darkfield photomicrographs of a trans-verse section through the GCT of Crocodylus niloticus, showing thedistribution of contralateral retinopetal neurons labeled with rhoda-mine beta-isothiocyanate (RITC) and the distribution of tyrosine hy-droxylase (TH) -ir somata (arrowheads) and fibers of passage. Notethat some retinopetal neurons are doubly labeled (arrows). For abbre-viation, see list. Scale bars � 100 �m.


Although few centrifugal visual neurons of crocodilesare TH-ir, a significant proportions of these are not onlyChAT-ir and NOS-ir but also immunonegative for 5-HT,GABA, and several neuropeptides. The neuroactive sub-stances used by these cells remains to be determined;possible neurotransmitters include aspartate, described inthe centrifugal visual fibers of turtles (Yaqub and Eldred,1991), and glutamate, identified in the centrifugal neu-rons and terminals of the pigeon (Rio, 1996) and lamprey(Rio et al., 2002b).

Many hypotheses, some contradictory, have been ad-vanced to explain the possible functions of the centrif-ugal visual system of vertebrates (Reperant et al., 1989;Uchiyama, 1989; Ward et al., 1991; Miceli et al., 1999,for reviews); however, those concerning the avian CVSare fairly consistent. This retino-tecto-isthmo-retinalloop, excitatory at all synapses (Li and Wang, 1999b;Miceli et al., 2000), may well be implicated in variousmechanisms linked to a selective increase of visual sen-sitivity related to several behaviors: (1) the detection oftargets in shadowed areas (Rogers and Miles, 1972), (2)attention during the search for food (Holden, 1990), (3)increased retinal sensitivity to a wide range of novel orbiologically important stimuli (Uchiyama, 1989; Miceliet al., 1995; Clarke et al., 1996), and (4) increasedstabilization of gaze to improve the precision with whichsmall objects are identified (Woodson et al., 1995). Ingeneral, these hypotheses all involve a dynamic processof selective enhancement or switching of visual atten-tion toward particular regions of the visual field.

Given that the plan of organization of the crocodilianCVS is comparable to that of birds, in particular theretino-tecto-isthmo-retinal loop (Ferguson et al., 1978),we may imagine that its function is also close to that ofbirds. It is evident that further investigations are calledfor to differentiate between the hypotheses that havebeen set forth to explain its function.

ACKNOWLEDGMENTS

The authors thank Prof. P. Grellier, UFR 63 CNRS-MNHN, for CLSM assistance, D. LeCren for skillful pho-tographic assistance, and B. Jay for help with computerdrawings.

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