Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: Distribution and comparison...

Calretinin Immunoreactivity in the Brainof the Zebrafish, Danio rerio:

Distribution and Comparison with SomeNeuropeptides and Neurotransmitter-Synthesizing Enzymes. II. Midbrain,Hindbrain, and Rostral Spinal Cord

ANTONIO CASTRO,1 MANUELA BECERRA,2 MARIA JESUS MANSO,1

AND RAMON ANADON2*1Department of Cell and Molecular Biology, Faculty of Sciences, University of A Coruna,

15071-A Coruna, Spain2Department of Ecology and Cell Biology, University of Santiago de Compostela,

15782-Santiago de Compostela, Spain

ABSTRACTThe distribution of calretinin (CR) in the brainstem and rostral spinal cord of the adult

zebrafish was studied by using immunocytochemical techniques. For analysis of some brainstemnuclei and regions, CR distribution was compared with that of complementary markers (cholineacetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y). The re-sults reveal that CR is a marker of various neuronal populations distributed throughout thebrainstem, including numerous cells in the optic tectum, torus semicircularis, secondary gusta-tory nucleus, reticular formation, somatomotor column, gustatory lobes, octavolateral area, andinferior olive, as well as of characteristic tracts of fibers and neuropil. These results indicate thatCR may prove useful for characterizing a number of neuronal subpopulations in zebrafish.Comparison of the distribution of CR observed in the brainstem of zebrafish with that reportedin an advanced teleost (the gray mullet) revealed a number of similarities, and also someinteresting differences. Our results indicate that many brainstem neuronal populations havemaintained the CR phenotype in widely divergent teleost lines, so CR studies may prove veryuseful for comparative analysis. J. Comp. Neurol. 494:792–814, 2006. © 2005 Wiley-Liss, Inc.

Indexing terms: calcium-binding proteins; immunohistochemistry; GAD; NPY; hindbrain;

zebrafish; Danio rerio

Calretinin (CR) is a cytosolic 29-kD calcium-bindingprotein (CaBP) of the EF-hand family, which was firstidentified by gene cloning from chick retina (Rogers,1987). The EF-hand calcium-binding proteins are selec-tive neuronal markers of distinct groups of neurons inboth the central and the peripheral nervous systems (Ce-lio, 1990; Rogers et al., 1990; Baimbridge et al., 1992;Resibois and Rogers, 1992; Andressen et al., 1993), includ-ing neuronal populations of the mammalian brainstem,such as the auditory nuclei and superior colliculus (Arai etal., 1991). Calcium-binding proteins (CaBPs) are involvedin intracellular calcium buffering, which contributes tomembrane properties and electrical activity of neurons(Miller, 1991; Lledo et al., 1992; Chard et al., 1993).

In teleosts, the presence of CR in optic fibers and insome neurons of the optic tectum of a cyprinid (Arevalo etal., 1995) and in different cells and fibers of the electrosen-

Grant sponsor: Spanish Education and Science Ministry; Grant number:BFU2004-05287/BFI.

*Correspondence to: Ramon Anadon, Department of Ecology and CellBiology, University of Santiago de Compostela, 15782-Santiago de Com-postela, Spain. E-mail: [email protected]

Received 9 February 2005; Revised 14 July 2005; Accepted 13 September2005

DOI 10.1002/cne.20843Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 494:792–814 (2006)

© 2005 WILEY-LISS, INC.

sory system of several mormyriforms (Friedman and Ka-wasaki, 1997) has been reported. A more general studyhas revealed a number of CR neuronal populations dis-tributed along the brain of an advanced teleost, includingvarious populations in brainstem centers (Dıaz-Regueiraand Anadon, 2000). A developmental study in trout indi-cates that CR immunoreactivity is expressed early in spe-cific forebrain populations that maintain this expressionthroughout development and adulthood (Castro et al.,2003).

The zebrafish is a model vertebrate for developmentalstudies, and numerous studies have dealt with early braindevelopment in this species. As far as we are aware, thedistribution of CR has not previously been studied in thebrain and spinal cord of developing or adult zebrafish.Here, we have analyzed the pattern of distribution of CRand other complementary neurochemical markers in thebrainstem of the adult zebrafish, with the aim of bettercharacterizing neuronal groups and centers. The organi-zation of forebrain systems expressing CR and some com-plementary markers [glutamic acid decarboxylase (GAD),tyrosine hydroxylase (TH), choline acetyltransferase(ChAT), neuropeptide Y (NPY), thyrotropin-releasing hor-mone (TRH), and galanin (GAL)] is dealt in a previouslypublished paper (Castro et al., 2006). Knowledge of thesepopulations may help in the comparison of the developing

and adult brain. Moreover, detailed knowledge of theadult brain is important for understanding changescaused in the brain by experimental manipulation, muta-tion, or genetic approaches.

MATERIALS AND METHODS

Animals

Adult zebrafish of both sexes (Danio rerio, Cyprinidae)were used in the present study (N � 27). All specimenswere obtained from a commercial supplier in A Coruna,Spain. Experimental procedures were approved by theanimal care and use committee of the University of ACoruna and conformed to the guidelines of the EuropeanCommunity.

CR immunohistochemistry

The methodology used in this study for CR is that re-ported elsewhere (see Castro et al., 2006). Briefly (forfurther details see Castro et al., 2006), the zebrafish weredeeply anesthetized with tricaine methane sulfonate(Sigma, St. Louis, MO) and perfused intracardially with4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4(PB). The brains were then removed from the skull, placedin the same fixative for 20 hours at 4°C, and left overnight

Abbreviations

AP area postremaATN anterior tuberal nucleusCB cerebellar bodyCC cerebellar crestCG central grayCIL central nucleus of the inferior lobeCN central nucleus of the torus semicircularisCON caudal octavolateralis nucleusCP central posterior thalamic nucleusDON descending octaval nucleusDP dorsal posterior thalamic nucleusDTN dorsal tegmental nucleusEG granular eminencefr fasciculus retroflexusGR granule cell layerHC horizontal commissureHY hypothalamusHYc caudal hypothalamusIII oculomotor nerveIIIn oculomotor nucleusIMR intermediate reticular formationIO inferior oliveIP interpeduncular nucleusIRF inferior reticular formationLIX glossopharyngeal lobeLL lateral lemniscusLR lateral recessLVII facial lobeLX vagal lobeMaO magnocellular octaval nucleusMB mammillary bodyMFN medial funicular nucleusML molecular layermlf medial longitudinal fascicleMON medial octavolateralis nucleusNDLI diffuse nucleus of the inferior hypothalamic lobeNI nucleus isthmiNLV lateral nucleus of the valvulaNmlf nucleus of the medial longitudinal fascicleON optic nerveOT optic tectumPGa anterior preglomerular nucleus

PGm medial preglomerular nucleusPHL posterior hypothalamic lobePL perilemniscal nucleusPOC posterior octaval nucleusPTN posterior tuberal nucleusPTO pretoral nucleusR CR-ir nucleus of the ventral rapheRC commissure of the raphe tractRF reticular formationSAC stratum album centraleSFGS stratum fibrosum et griseum superficialeSGC stratum griseum centraleSGN secondary gustatory/visceral nucleusSgt secondary gustatory tractSL superficial (periventricular) layer of the torus semicircu-

larisSM stratum marginaleSO stratum opticumSPV stratum periventriculareSR superior reticular formationT tangential octaval nucleusTbs bulbospinal tractTC transverse commissureTel telencephalonTGN tertiary gustatory nucleusTgt tertiary gustatory tractTL torus longitudinalisTLA torus lateralisTPM pretectomammillary tract and nucleusTS torus semicircularisV fourth ventricleVC cerebellar valvulaVdr trigeminal descending rootVH ventral hornVIII octaval ganglion and nerveVIIm facial motor nucleusVIIs facial sensory rootVL ventrolateral midbrain reticular formationVn trigeminal nerveX vagal nerveXm vagal motor nucleusXr vagal reticular region

793CALRETININ IN THE ZEBRAFISH BRAINSTEM

Fig. 1. A–P: Schematic drawings of transverse sections of thebrainstem and spinal cord of zebrafish showing, at right, the distri-bution of CR-immunoreactive (CR-ir) perikarya (dots) and fields richin CR-ir processes (shaded areas). Differences in process richness arerepresented in different grays (more dark, more rich). Some conspic-uously CR-ir fiber bundles, tracts and commissures are also indicatedby dashes or shading. The main nuclei and regions are indicatedschematically at left; the nomenclature used for brain structures was

based mostly on the zebrafish brain atlas of Wullimann et al. (1996).For abbreviations see list. Arrows in A–H point to the CR-ir raphetract decussating in the postoptic region. Stars in A–C indicate themesencephalic ventricle. Asterisks in D–N indicate the fourth ventri-cle. Boxed areas in H–J,L,M,O,P indicate the location of some photo-graphs. The location of transverse sections is indicated by lines in theinset representing a lateral view of the brain. Scale bars � 250 �m inE (applies to A–E), P (applies to F–P).

Figure 1 (Continued)


in a solution of 30% sucrose in PB. Afterward, the brainswere serially cut into transversal and sagittal sections ona cryostat or a vibratome.

The immunocytochemical analysis was carried out byusing the peroxidase-antiperoxidase method (PAP). Cryo-stat sections were mounted on subbed slides and wereblocked with 10% normal goat serum (Dako, Glostrup,Denmark) and later with a rabbit CR antiserum (SWant,Bellinzona, Switzerland; No. 7699/4, lot 18299) diluted1:1,000 in phosphate-buffered saline (PBS). After beingrinsed with 0.01 M PBS, pH 7.4, sections were treated for1 hour with goat anti-rabbit immunoglobulin G (1:50;Dako; No. Z0421) and rinsed several times in PBS. Afterincubation for 1 hour in rabbit PAP (1:200; Dako; No.Z0113) and successive washes in PBS and in 0.05 M Tris-HCl buffer at pH 7.6, sections were developed with 0.06%3,3�-diaminobenzidine hydrochloride (DAB; Sigma) and0.005% H2O2 in Tris-HCl for 5–10 minutes. Finally, sec-tions were rinsed in Tris-HCl, dehydrated through alco-hol, cleared with xylene, and coverslipped with Eukittmounting medium. Free-floating vibratome sections wereprocessed similarly, but the time of incubation of the dif-ferent antisera was doubled. The CR antibody was raisedagainst recombinant human CR. Different controls wereperformed 1) by incubating sections similarly but withoutthe CR antiserum and 2) by incubation with the CR anti-serum after preabsorption with 0.172 �M recombinanthuman CR (SWant). In both cases, no staining was ob-served. The primary antibody was also tested in brainextracts by Western blotting. In these blots, the CR anti-body stained a single band of about 28–29 kD (see Castroet al., 2006).

Other methods

As complementary markers for better characterizationof brainstem regions, we also used series of transversesections immunostained with rabbit polyclonal antibodiesto synthetic porcine NPY (Sigma; No. N-9528; lot047H4826; dilution 1:1,000), recombinant feline GAD(Chemicon; No. AB5992; lot 24030212; dilution 1:1,000),rat pheochromocytoma TH (Chemicon; No. AB152; lots22030650 and 22040492; dilution 1:1,000), and goat poly-clonal antibody to human placental ChAT (Chemicon; No.AB144P; lot 25030685; dilution 1:100). The protocols andcontrols used were as published elsewhere for trout (Cas-tro et al., 1999; Perez et al., 2000; Anadon et al., 2002) anddogfish (Sueiro et al., 2004).

For topographic purposes, we used Nissl-stained trans-verse series of adult brain. For Nissl staining, two adultzebrafish were fixed in Bouin’s fluid. Brains were embed-ded in paraffin wax and cut serially on a rotary microtome(10–12 �m in thickness). Dewaxed sections were rehy-drated and stained in 0.62 mM cresyl violet (Analema,Vigo, Spain), 24 mM sodium acetate, and 49.9 mM aceticacid for 30 minutes. Color differentiation was performedin 96% ethanol for 5 minutes, and then sections weredehydrated and mounted.

Photomicrographs were taken with an Olympus micro-scope equipped with a color digital camera (DP12; Olym-pus Co., Tokyo, Japan), converted to gray scale, and ad-justed for contrast and brightness with Corel Photoshop(Corel, Ottawa, Ontario, Canada). Photomontage and let-tering were done with Corel Draw (Corel). The nomencla-ture used in this study is based on that of the zebrafishbrain atlas of Wullimann et al. (1996).

RESULTS

The CR antibody used in this study is well characterizedand has been tested by Western blotting in brain extractsof teleosts (Dıaz-Regueira and Anadon, 2000; Castro et al.,2003). Moreover, Western blotting of protein extracts ofzebrafish brain reveals a single band of about 28–29 kD,an MW slightly lower than that revealed in trout and ratbrain extracts (see Fig. 1 in Castro et al., 2006). Incuba-tion of the sections without the primary antibody or withthe primary antibody preabsorbed with human CR pro-duced no staining of these structures, indicating that thestaining is specific of CR.

Immunocytochemistry with the anti-CR antibody re-vealed neurons and fibers distributed in the brainstemand rostral spinal cord. Figure 1 shows schematically thedistribution of CR-immunoreactive (-ir) structures ob-served in transverse sections of the zebrafish brainstem.Results obtained with ChAT immunohistochemistry arelargely coincident with those recently reported for ze-brafish by Clemente et al. (2004) and Mueller et al. (2004),and they will be mentioned only in the context of CR-irpopulations. The distribution of TH in the zebrafish brain-stem has also been described in several studies (see Ma,1997; Kaslin and Panula, 2001), and here this marker isused mainly for topological reference for some CR popula-tions. The brain distribution of other complementarymarkers (GAD, NPY) has not been studied in zebrafish.However, these markers are reported here in relation tosome CR-ir populations, and a more general description isbeyond the scope of the present study.

Midbrain

Optic tectum and torus longitudinalis. The layeringof the optic tectum of adult zebrafish is similar to that ofthe goldfish (see Meek and Schellart, 1978), so here weadopt Meek and Schellart’s nomenclature of layers andcells. From the ependyma to the outer surface, the follow-ing strata are considered: the stratum periventriculare(SPV) in the inner region, the stratum album centrale(SAC), the stratum griseum centrale (SGC), the stratumfibrosum et griseum superficiale (SFGS), the stratum op-ticum (SO), and the stratum marginale (SM; Figs. 1A–E,2A). The optic tectum contains a number of CR-ir struc-tures, both neuronal perikarya and fibers (Fig. 2A–G). Alarge proportion (roughly about 35%) of the smallperikarya of the SPV is CR-ir (Fig. 2A). These cells have aradial process extending to the SO, which gives the inter-mediate layers a fine striated appearance in thick sec-tions. Scarce CR-ir cells were observed in the SAC, mostlyabove the fiber bundles coursing horizontally in this layer.A few of these cells had a horizontal orientation. In theSGC (mostly in its outer border), CR-ir cells were rathermore numerous (Fig. 2A,G). These bipolar cells also ex-hibited radial processes. A few CR-ir bipolar cells withshort apical dendrites were found in the SFGS (Fig. 2E).In the superficial SO, there were also scattered globularCR-ir perikarya (Fig. 2B), probably corresponding to Meekand Schellart’s type III cells, and, in the deep zone of theSO, bipolar CR-ir cells were also observed (Fig. 2D; seebelow).

The stratum marginale was devoid of CR-ir structures,with the exception of a few pear-shaped perikarya in therostralmost region of the tectum (Fig. 2C). The SO showedabundant thick CR-ir fibers (Fig. 2A,B,D). These formed

796 A. CASTRO ET AL.

Fig. 2. Vertical sections through the optic tectum showing CR-immunoreactive neurons. A: Thin transverse section showing CR-irperikarya in different layers of the tectum. B: Detail of the stratumopticum showing CR-ir perikarya associated with CR-ir optic fibers inexternal (solid arrow) or internal (open arrow) location. C: Pear-shaped CR-ir perikaryon (arrow) located in the stratum marginale inthe rostral region of the tectum. D: Bipolar CR-ir cell located below

the stratum opticum. E: CR-ir bipolar cell of the stratum fibrosum etgriseum superficiale. F: Pear-shaped CR-ir neurons of the stratumperiventriculare exhibiting long apical processes. G: Bipolar CR-ir cellof the stratum griseum centrale. Note that the apical dendrite givesrise to a horizontal axon. For abbreviations see list. Scale bars � 50�m in A; 12.5 �m in B,D–G; 25 �m in C.


two sublayers, one thin superficial layer and a thickerdeep layer (pars profunda) with more densely packed andrather thicker CR-ir fibers. These two sublayers were sep-arated by a CR-negative band in the rostral tectum butwere directly apposed in most parts. In the rostral tectum,the superficial part of the SO is continuous with fiberspassing through and around the PSP, whereas the parsprofunda is continuous with the major optic tracts. The

SFGS showed numerous thin CR-ir processes, but thelayer was clearly lighter than the SO (Fig. 2A). The SGCshowed thin bouton-like processes, mostly in its outerborder, in addition to the radial CR-ir processes men-tioned above.

The optic tectum showed numerous GAD-ir cells andfibers (Fig. 3A). Rather widely spaced GAD-ir cells with anouter radial process were observed in the SPV (Fig.

Fig. 3. Vertical sections through the optic tectum showing struc-tures immunoreactive to GAD (A,D–G), ChAT (B), and TH (C). A: Sec-tion through the lateral region of the optic tectum showing the generaldistribution of GAD-ir cells (arrows) and neuropil. B: Similar sectionshowing the distribution of ChAT-ir neurons and neuropil. C: Verticalsection of the dorsomedial optic tectum showing the distribution ofTH-ir fibers. D: Section showing GAD-ir neurons in the limit between

the stratum marginale (SM) and the stratum opticum. Note scatteredGAD-ir boutons in the SM. E: GAD-ir neurons located in the stratumgriseum centrale. F: Large pear-shaped GAD-ir neuron of the stratumperiventriculare showing a rather thick apical dendrite. G: FaintGAD-ir small neurons (thin arrows) located in the limit between thestratum periventriculare and the stratum album centrale. Scalebars � 25 �m in A–C; 12.5 �m in D–G.


3A,F,G). Some fine GAD-ir boutons were also observed inthis layer among negative perikarya. Small GAD-ir neu-rons were also found in the SGC and SFGS, although inthese layers the high density of GAD-ir boutons makes itdifficult to distinguish them (Fig. 3A,E). In the superficialpart of the SO, among the GAD-negative fiber tracts, thereare numerous small GAD-ir cells (Fig. 3A,D). The SO parsprofunda was filled with a large number of GAD-ir pro-cesses (Fig. 3A) and a few GAD-ir cells. In the stratummarginale, there are numerous small GAD-ir boutons(Fig. 3A,D), though at a much lower density than in thecentral layers of the tectum. These central layers (SAC,SGC, SFGS, and SO pars profunda) show very numerousGAD-ir terminals but exhibit a layered appearance be-cause of differences in density at the transition betweenlayers (Fig. 3A). The fiber layer of the SAC likewiseshowed scarce GAD-ir processes.

Our ChAT results confirm that the zebrafish optic tec-tum contains radial ChAT-ir neurons in the SPV (Clem-ente et al., 2004; Mueller et al., 2004). The dendrites ofthese cells branch and form horizontal plexuses betweenthe SAC and the SGC, between the SGC and the SFGS,and along most of the SFGS and SO pars profunda (Fig.3B). Thin beaded ChAT-ir fibers course in the SAC outsidethe tectum, and contribute to a rich ventrolateral reticulartegmental field of coarse ChAT-ir boutons. The tectumalso shows small NPY-ir cells in the SPV, but their radialprocesses are scarcely visible; instead, a number of coarsebeaded NPY-ir fibers course through the SAC, SGC,SFGS, and deep SO without clear lamination or radialorientation. A few NPY-ir fibers also run in the SM andthe SPV.

Numerous TH-ir fibers were observed in the tectum(Fig. 3C). These fibers enter the tectum through the SACfrom the pretectal TH-ir population and distribute widelyin two bands: on both sides of the SAC (in the outer partof the SPV and inner SGC) and in the SFGS. There is adramatic difference between a dorsomedial band showingrich TH-ir innervation and the more extensive ventrolat-eral and caudoventral region of the tectum, which practi-cally lacks TH-ir innervation. The transition betweenthese two tectal zones is rather abrupt.

No CR-ir structures were observed in the torus longitu-dinalis. The torus longitudinalis showed lines of coarseGAD-ir boutons located among groups of GAD-negativegranule cells, a pattern reminiscent of that observed in themammillary body and the cerebellar granular layer. PaleGAD-ir neurons are also observed in the torus, mainly indorsal regions near the optic tectum. Some NPY-ir fibersalso course in the torus longitudinalis. No ChAT-ir orTH-ir fibers were observed.

Torus semicircularis. The torus semicircularis showsa number of faintly to moderately CR-ir cells (Figs. 1B–D,4A). In periventricular regions, the CR-ir cells are rathersmall, whereas, in a central band extending through thecentral and ventrolateral parts of Wullimann et al. (1996),the CR-ir cells are clearly larger. The torus semicircularisshows a neuropil with thin CR-ir processes. Bundles withsome CR-ir fibers cross the torus semicircularis on theirway between the tectum and the tegmentum. The torussemicircularis shows numerous GAD-ir boutons but lacksGAD-ir perikarya (Fig. 4B). The torus semicircularis isinnervated by rather numerous NPY-ir fibers. In theperiventricular tegmentum located medial to the torus,

there are large NPY-ir neurons in the nucleus referred toby Wullimann et al. (1996) as Edinger-Westphal nucleus.

Tegmentum. The midbrain tegmentum contains theoculomotor nucleus, and wide lateral and ventral regionsthat show ill-defined and poorly characterized neuronalpopulations. CR immunohistochemistry reveals neuronsmainly in the oculomotor nucleus and in the lateral teg-mentum (Fig. 1C,D).

The oculomotor nucleus forms a long arch over the me-dial longitudinal fascicle (mlf), as seen with ChAT immu-nocytochemistry (Fig. 4C). The oculomotor neurons arefaintly CR-ir in some parts (Fig. 4D,E). Of more interest isthat the nucleus shows clearly different parts with regardto the pattern of innervation by CR-ir fibers (Fig. 4D–H).The ventromedial part of the nucleus receives very thickCR-ir fibers that form calyx-like terminals on single cells(Fig. 4E,H). By contrast, the dorsolateral part shows anumber of small CR-ir boutons around cell bodies (Fig.4D–G). Caudal to the oculomotor nucleus, in a similarposition over the mlf, there is a smaller group of ChAT-irneurons that form the trochlear nucleus, topologically lo-cated in the rostral isthmus. These cells are also CR-irbut, unlike the case in the oculomotor nucleus, no specialpattern of innervation of the nucleus by CR-ir fibers wasobserved.

The tegmentum is traversed from caudodorsally to ros-troventrally by conspicuous CR-ir bundles of the tertiarygustatory tract (Fig. 1C,D), which originate from the sec-ondary gustatory nucleus, and a thick bundle (the laterallemniscus) originating from the medullary lateral lineregion (medial octavolateralis nucleus) and ending in thetorus semicircularis. Only a few CR-ir fibers course in thelateral lemniscus. At the level of the third nerve exit, thereis a conspicuous commissure bearing numerous CR-ir fi-bers close to the meninges, the transverse commissure(Fig. 1C,D). For the description of the tegmental CR-irpopulations, these tracts and commissure are here used astopographical references. In the tegmentum, numerousCR-ir cells were observed scattered laterally to the ter-tiary gustatory tract and dorsolaterally to the lateral lem-niscus in the region of the perilemniscal nucleus (Fig.1C,D). This CR-ir population reaches caudally to the levelof the secondary gustatory nucleus. A small group ofmedium-sized CR-ir multipolar cells is located in the ros-tral ventrolateral tegmentum lateral to the tertiary gus-tatory tract and ventral to the lateral lemniscus (Fig. 1C).These cells probably correspond to the ventrolateral retic-ular formation.

Isthmus and rhombencephalon

Interpeduncular nucleus. The interpeduncular nu-cleus shows some small CR-ir cells, mainly rostral ordorsal to the neuropil or in the intermediate region be-tween the dorsal and the ventral neuropil (Figs. 1D, 4I).The interpeduncular neuropil is rich in GAD-ir boutons,and GAD-ir cells are found over the nucleus or in theintermediate region between the dorsal and the ventralneuropil (Fig. 4J,K). NPY-ir fibers are rather numerous inthe interpeduncular nucleus.

Secondary gustatory/visceral nucleus, lateral nu-

cleus of the valvula, and nucleus isthmi. The second-ary gustatory/visceral nucleus (SGN) is a prominent nu-cleus located in the rostral isthmus close to the nucleusisthmi and the cerebellar valvula and in contact rostrallywith the lateral nucleus of the valvula (Fig. 5A,B). A broad


Figure 4


SGN commissure joins the nuclei of both sides over thefourth ventricle (Fig. 5A). The secondary gustatory/visceral nucleus showed numerous CR-ir cells, distributedin a large dorsal locus and a smaller ventrorostral hori-zontal lamina with closely grouped strongly CR-ir smallcells (Figs. 1D,E, 5C–F). The SGN shows numerous CR-irprocesses in the central neuropil (Fig. 5C,F), and the com-missure of the SGN also exhibits strongly CR-ir fibers.From the SGN, bundles of CR-ir fibers course rostroven-trally toward the inferior hypothalamic lobe (Figs. 1C,D,5E). The ventral lamina of the SGN also exhibits numer-ous ChAT-ir cells (Fig. 5G), in general with location sim-ilar to that of the ventrorostral lamina of strongly CR-ircells. This part of the nucleus sends bundles of ChAT-irfibers toward the inferior hypothalamic lobe, but the SGNcommissure was ChAT negative. No ChAT-ir fibers wereobserved in the cerebellar valvula or the corpus, suggest-ing that these ChAT-ir cells do not project to the cerebel-lum (see Discussion). The SGN is innervated by thin orvery thin GAD-ir boutons, except for the rostral lamina ofstrongly CR-ir cells, which has a small, dense core ofrather coarse GAD-ir boutons (Fig. 5H). NPY-ir fibers arenumerous in the ventrolateral region of the SGN, and afew enter the SGN commissure, but they are lacking inmost regions of the SGN. Some NPY-ir perikarya areobserved caudal to the SGN in the region of the locuscoeruleus.

The lateral nucleus of the valvula (NLV) appears as awedge-shaped lamina of small cells located dorsorostrallyto the secondary gustatory nucleus. The NLV enlargesrostrally to the tertiary gustatory tract, whose exit servesas the approximate limit with the SGN. The NLV consistsof CR-negative and ChAT-negative small cells but receivesfinely varicose CR-ir fibers (Fig. 5E) and also some cup-shaped ChAT-ir fibers. GAD immunohistochemistry al-lows for differentiating this nucleus from the adjacentgranular layer of the cerebellar valvula and the secondarygustatory nucleus, because it has a very scarce GAD-irinnervation. Instead, the valvula shows the characteristicbeaded mossy fibers covered by GAD-ir boutons (see belowunder Cerebellum), whereas the secondary gustatory nu-

cleus has dorsal parts with thin GAD-ir fibers and a ven-trorostral part innervated by coarse GAD-ir boutons (Fig.5H). Nissl staining with cresyl violet also indicates thatthe NLV consists of numerous small granule cells withscarce cytoplasm associated with a conspicuous tract en-tering the valvula cerebelli. These cells are smaller andexhibit much less cytoplasm than those of the rostral partof the SGN, which supports the distinction of these nucleibased on neurochemical differences.

The nucleus isthmi is an ovoid nucleus in transversesection, characterized by its location just lateral to thesecondary gustatory nucleus and its rim of moderatelyChAT-ir cells around a central neuropil (Fig. 5A,C,F–H).It is traversed by the trochlear nerve root. The cells of thenucleus are CR negative (Fig. 5A). This nucleus is clearlyidentifiable by GAD immunocytochemistry, showing anumber of coarse GAD-ir boutons in the neuropil (Fig.5H). No NPY-ir fibers are observed in this nucleus, al-though they are abundant in neighbor regions.

Cerebellum. CR immunocytochemistry revealed someCR-ir structures in the zebrafish cerebellum. Bundles ofCR-ir fibers enter the corpus cerebelli through the cere-bellar peduncles and ascend to form a plexus below thePurkinje cell layer. From this plexus, isolated CR-ir fibersascend in the basal one-third of the molecular layer, givingrise to compact, claw-shaped bunches of boutons (Fig.6A,B). These claw-shaped endings were observed through-out the molecular layer and probably represent climbingfibers. However, whether they contact Purkinje-cell den-drites or some other type of cell in the molecular layer isnot known. The molecular layer has very faint GAD-irsmall cells (stellate cells) and rather regularly spacedGAD-ir thin, beaded fibers coursing in parallel, generallyaligned in the transversal plane, i.e., in the same directionas the parallel fibers (Fig. 6C). We did not find any GAD-irstructures showing any sort of correspondence to theCR-ir “claws.”

The granular layer contains strongly CR-ir small cells ofgranule cell appearance, singly or in small groups (Fig.6D), although the molecular layer does not exhibit CR-irparallel fibers. In the caudal part of the corpus cerebelli,there were large, pale CR-ir neurons with long dendritesentering the molecular layer caudal to the cerebellar pe-duncle. A few such cells, showing rostrocaudal orientation,were also observed in the ventral part of the valvulacerebelli (Fig. 6E). The granular layer of the cerebellumshows numerous lines of small, strongly GAD-ir boutonscoursing among the compact cords of GAD-negative gran-ule cells, delineating the mossy fibers. In some regions ofthe corpus (caudal, rostral) and valvula cerebelli (ventro-medial), dense boutons delineate the perikarya and thickdendritic stems of rather large neurons (eurydendroidcells), located in the granular layer near the Purkinje cells(Fig. 6F). The Purkinje cell perikarya are only faintly orvery faintly GAD-ir and are contacted by very thin GAD-irboutons. Among Purkinje cells or in the neighboring gran-ular layer, there are a few neurons showing a scantGAD-ir cytoplasm that might correspond with Golgi cells.The nuclear size of these neurons is intermediate betweenthe sizes of Purkinje cells and granule cells. A few NPY-irfibers are observed in the granular eminences.

Motor systems. The visceromotor neurons of the tri-geminal, facial, glossopharyngeal, and vagal nuclei areeasily distinguished, because they show strong ChAT im-munoreactivity, in agreement with previous reports for

Fig. 4. Photomicrographs of sagittal (A,H) and transverse (B–G,I–K) sections of the midbrain-isthmus showing the distribution ofCR-ir (A,D–I), ChAT-ir (C), and GAD-ir (B,J,K) structures. A: Sectionof the torus semicircularis showing CR-ir neurons in the superficiallayer (SL) and central nucleus (CN). Upper left corner, optic tectum.B: Distribution of GAD-ir boutons in the superficial and central partof the torus semicircularis. C: Distribution of ChAT-ir motoneurons inthe oculomotor nucleus. Note the presence of oculomotoneurons inboth medial and dorsolateral (arrow) parts. Asterisk, medial longitu-dinal fascicle; star, midbrain ventricle. D–F: Thick sections in rostro-caudal progression showing the region of the oculomotor nucleusreceiving large cup-shaped terminals (open arrows in D,E) originatingfrom a bundle of thick CR-ir fibers (arrow in F). The solid arrows inD,E point to the dorsolateral region receiving thin CR-ir fibers. As-terisk in D, medial longitudinal fascicle; star in E, midbrain ventricle.G: Detail of the branched thin CR-ir fibers ending in the dorsolateralpart of the oculomotor nucleus (arrow). H: Thick CR-ir fibers forminglarge cup-shaped terminals around oculomotoneurons (arrows).I: Section of the interpeduncular nucleus showing a few faint CR-irneurons (arrows). J: Section of the interpeduncular nucleus showingthe rich GAD-ir neuropil. The arrow points to a GAD-ir neuron.Asterisk, fasciculus retroflexus. K: Detail of a GAD-ir neuron in theinterpeduncular nucleus (arrow). Asterisk, fasciculus retroflexus.Scale bars � 50 �m in A–F,I–J; 25 �m in G,H; 10 �m in K.


Figure 5

zebrafish (Clemente et al., 2004; Mueller et al., 2004).However, these nuclei do not contain CR-ir motoneurons,although a few intensely CR-ir reticular neurons appearintermingled with CR-negative motoneurons.

The zebrafish abducens nucleus is located rather ven-trally in the region just caudal to the octaval nerve en-trance. In the abducens nucleus, strongly CR-ir neuronswere observed, although whether they were interoculomo-tor neurons or motoneurons was not determined.

Reticular formation. The rhombencephalic reticularformation contained numerous CR-ir neurons distributedat isthmotrigeminal (rostral), octaval (middle), and caudallevels (Fig. 1F–M). Most large CR-ir reticular neurons arelocated at different heights (dorsal, ventral) in a medialband parallel to the mlf, to which many of these cellscontribute, but in the rostral rhombencephalon there areCR-ir groups of large cells located rather laterally (Fig.7A–E). In sagittal sections, the large medial CR-ir reticu-lar neurons appear segregated in segmental groups. Manyof the larger CR-ir reticular cells can be recognized asreticulospinal neurons by comparison with the detaileddescriptions based on tract-tracing studies in larval andadult zebrafish (Kimmel et al., 1982, 1985; Metcalfe et al.,1986; Lee and Eaton, 1991). In accordance with this iden-tification, more than 90% of the thick fibers of the mediallongitudinal fascicle along the rhombencephalon and thespinal cord, in both its dorsal and its ventral parts, arestrongly CR-ir. The number of these axons is about 70–75on each side of the medial longitudinal fascicle. However,the Mauthner cell and its axon were CR negative. TheMauthner cell axon cap is covered by numerous coarseCR-ir fibers (Fig. 7F,G), and thin CR-ir fibers appear alsoto contact the Mauthner cell perikaryon or dendrites. Inthe dorsal part of the medial longitudinal fascicle, thereare one or two CR-negative axons about a one-third of the

diameter of the Mauthner axon, which could correspond toaxons of the Mauthner cell serial homologues (Lee andEaton, 1991).

GAD immunocytochemistry shows that the Mauthnercell perikaryon and the thick lateral dendrite are“cobblestone-covered” with large GAD-ir boutons (Fig.7H). However, the other reticular cells have sparserGAD-ir innervation, and only small GAD-ir boutons areobserved on their perikarya and dendrites. Small GAD-irneurons are observed in the medial reticular formationlaterally to the medial longitudinal fascicle.

In addition to the putative large reticulospinal cells, thereticular formation exhibits other CR-ir cells, large andsmall, located at medial, intermediate, or lateral levels intransverse sections (Fig. 7A,C,D). In sagittal sections, thesmall cells lack a clear segmental arrangement.

Central gray and raphe region. At isthmic-pretrigeminal levels, the central gray consists of small,pear-shaped neurons that exhibit strong CR immunoreac-tivity located over the mlf (Figs. 1F, 7A,I). These cellsexhibit processes coursing toward the medial longitudinalfascicle.

In the trigeminal region, there is a small but conspicu-ous group of CR-ir small neurons located near the ventralsurface (Fig. 7J–M). This nucleus is considered here as apart of the raphe complex. Most of these cells are locatedin the ventral midline (Fig. 7J,K), but rostrally some cellsaccompany a conspicuous paired CR-ir tract that coursesalong the basal midbrain to the inferior hypothalamiclobes and then to the postoptic region, where it decussates(Figs. 1A–G, 7L,M). Apparently, the only target of thisCR-ir fascicle is a dorsomedial region of the nucleus of thelateral recess (see Castro et al., 2006). Here and in apreviously published paper (Castro et al., 2006), this tractis referred to as the raphe tract.

Octavolateral region. At the octaval nerve entrancelevel, there are large CR-ir cells that send long brancheddendrites dorsalward, reaching the medial octavolateralisnucleus and occasionally the cerebellar crest that covers it(Figs. 1H,I, 8A). These cells correspond to the magnocel-lular octaval nucleus. Characteristically, the perikarya ofthese magnocellular cells are covered by numerous, coarseGAD-ir boutons. Just at the entrance of the octaval nerve,there are smaller monopolar CR-ir cells distributed in twoareas, some intermingled with primary octaval fibers (dor-sal part of the tangential nucleus; Tello, 1909) and othersin a group located ventromedially to the nerve entrance(ventral part of the tangential nucleus; Tello, 1909; Figs.1I, 8B). The axons of the cells of the dorsal part coursedorsomedially toward the mlf, whereas those of the ven-tral tangential nucleus decussate in a conspicuous com-missure and give rise to a crossed descending vestibulartract. Small CR-ir cells are also observed in the descend-ing octaval nucleus, although in small numbers (Figs.1I–K, 8C,F). No CR-ir cells were observed in the regioncorresponding to the secondary octaval population of thegoldfish (McCormick and Hernandez, 1996), i.e., medial tothe sensory root of the facial nerve and ventral to thecerebellar crest.

CR-ir octaval nerve fibers enter the ventral octavolate-ralis nucleus and give rise to a number of coarsely beadedfibers coursing to the different parts of the nucleus (themagnocellular, descending, ventral, and superior octavalnuclei) as well as to the cerebellar granular eminences, asseen in sagittal sections (Fig. 8D–F). The anterior and

Fig. 5. Photomicrographs of sections through the secondary gus-tatory nucleus (SGN) showing the organization in Nissl stains (A,B)and the immunoreactivity to CR (C–F), ChAT (G), and GAD (H).A,B: Sections of the SGN (solid single arrows) at the level of the SGNcommissure (A) and a rostral SGN level (B) showing close topograph-ical relationship with the valvula (star) and nucleus isthmi (arrow-heads). Double arrows point to the ventral celled region of the SGN. InA, the open arrow points to cells of the superior reticular nucleus. InB, the angled arrow points to the most caudal region of the nucleus ofthe valvula. C–E: Thick sections of the SGN showing numerous smallCR-ir neurons in the dorsal parts (straight single arrows) and thedensely celled ventral region (double arrows). The star indicates thecerebellar valvula. In C,D, the arrowheads indicate the nucleusisthmi. In E, note the loops of bundles SGN-hypothalamic tertiarygustatory tract (wide arrows) and CR-ir fiber terminals (angled ar-rows) in the caudal part of the nucleus lateralis valvulae. F: Thinsection of SGN showing the layered appearance of the neuropil.Straight arrows point to the CR-ir cells of the dorsal part, the doublearrow point to the CR-ir cells of the ventral part; arrowheads point tothe nucleus isthmi; the curved arrow points to large CR-ir neurons ofthe region of the locus coeruleus, and the open arrow to the trochlearnerve root. Star, fourth ventricle; Asterisk, lateral lemniscus. G: Sec-tion showing strongly ChAT-ir neurons in ventral and central regionsof the SGN (solid arrows) and faintly stained neurons in the nucleusisthmi (arrowheads). The open arrow points to a ChAT-ir reticularneuron. Asterisk, lateral lemniscus. H: Section showing the differentpatterns of GABA immunoreactivity in the nucleus isthmi (arrow-heads), which shows coarse boutons, and in the SGN, which shows asparsely innervated part (solid arrows) and a conspicuous GAD-irband in the densely celled ventromedial region (open arrow). Asterisk,lateral lemniscus. Scale bars � 50 �m.


posterior lateral line nerves exhibit only weak CR-ir fibersthat course in the lateral region of the medial octavolate-ralis nucleus. The cerebellar crest overlying the medialoctavolateralis nucleus lacks CR-ir structures, except foroccasional octaval neuron dendrites (Fig. 8A). The medialoctavolateralis nucleus contains a large number of GAD-irboutons, whereas the cerebellar crest shows rather sparseGAD-ir innervation. The octaval ganglion and nerve haveintensely CR-ir bipolar ganglion cells and nerve fibers(Figs. 1H,I, 8G).

Viscerosensory lobes. As in other cyprinids, theadult zebrafish possesses well-developed viscerosensorylobes of the facial, glossopharyngeal, and vagal nerves(Fig. 1J–M). In these lobes, numerous small cells, locatedeither in the border or more sparsely inside the lobes, wereCR-ir (Fig. 9A,D). However, the CR-negative small cellsappear at least as numerous as the CR-ir neurons. LargerCR-ir cells are also observed in ventral regions of thelobes. In addition, CR-ir fibers are present in the viscer-osensory nerve roots, giving rise to CR-ir plexuses in theneuropil below the outer cell mantle (Fig. 9A,D).

Small GAD-ir perikarya can be observed in the superfi-cial layer of the viscerosensory lobes. These lobes containvery large numbers of small GAD-ir boutons that arerather homogeneously distributed in the central neuropiland cell region (Fig. 9B). The vagal lobe also containsnumerous NPY-ir fibers, although these show a layeredpattern (Fig. 9C). A high density of terminals is observedin a thin layer below the neuronal cortex, which is fol-lowed by a large neuropil area with sparse NPY-ir struc-tures and a deep region with rather abundant NPY-irfibers. The facial lobe shows scattered NPY-ir fibers. Inaddition, some medium-sized NPY-ir neurons were ob-served associated with the glossopharyngeal and vagalmotor nucleus.

Area postrema. The zebrafish area postrema (AP) islocated in the dorsal midline of the obex, caudally to thevagal lobes (Figs. 1O, 9E–G). The AP contains highlyCR-ir small neurons, most with their perikarya closelyassociated with the dorsal meninges, toward which theysend short branched processes (Fig. 9E). In sagittal sec-tions, some of these cells showed a bipolar appearance,with thin axonal processes coursing ventrally. TH-ir neu-rons were also present in the AP with a location similar tothe CR-ir cells (Fig. 9F). In striking contrast with theviscerosensory lobes, the AP lacks both GAD-ir andNPY-ir perikarya. However, NPY-ir fibers were ratherabundant in this area (Fig. 9G).

Inferior olive. The inferior olive showed a number ofmoderately CR-ir cells (Figs. 1M, 9H). This rather com-pact nucleus was located close to the ventral surface in thecaudal medulla oblongata near the transition to the spinalcord. It originates CR-ir fibers that cross the midline andascend in the contralateral side toward the cerebellum.The inferior olive of zebrafish shows moderate innervationby GAD-ir boutons (Fig. 9I). A few GAD-ir perikarya werealso observed just dorsal to the inferior olive (Fig. 9I).

Rostral spinal cord

In the spinal cord, motoneurons, including theirperikarya, dendrites, and axons leaving the cord andcoursing in the ventral roots, were intensely CR-ir (Figs.1O,P, 9J,K). The medial longitudinal fascicle also showeda number of thick CR-ir axons, although the giant Mau-thner axons were CR negative (Fig. 9J). Before acquiring

their characteristic thick diameter, the initial segments ofthe large dorsal motoneurons passed close to (contacting?)the Mauthner axon (Fig. 9K). In the lateral funiculus,thick CR-ir axons were also observed. Dorsal horn spinalregions practically lacked CR-ir cells and processes.

DISCUSSION

In the present study, the distribution of CR in the brain-stem and rostral spinal cord of the adult zebrafish wasinvestigated with immunocytochemical techniques. Ourresults reveal that this species presents various CR neu-ronal populations distributed throughout the brainstem.In addition, CR immunocytochemistry reveals a numberof CR-ir fibers and terminals. Various centers in the mid-brain, cerebellum, and rhombencephalon contain one orseveral CR-ir neuron types. Moreover, comparison of thedistribution of CR observed in the brainstem of zebrafish(present results) with that reported for an advanced te-leost (Dıaz-Regueira and Anadon, 2000) reveals a numberof similarities, and also some interesting differences. Thissuggests that some aspects of the CR organization havebeen maintained in different teleost evolutionary lines,which raises the possibility that CR studies may be usefulfor comparative analyses.

Midbrain

Our results reveal several CR-ir cells and CR-ir fibersystems in the zebrafish optic tectum, exhibiting a layeredorganization. In combination with other markers, theyprovide further information on the tectum organization.CR-ir cells are found mainly in the stratum periventricu-lare (SPV), but also in more superficial layers. The num-ber of CR-ir cells in the SPV is much higher than that ofChAT-ir, GAD-ir, or NPY-ir neurons in this layer. Al-though most of these SPV cells probably correspond to thetype XIV cells of Meek and Schellart (1978), our immuno-cytochemical results reveal different lamination patternsof the immunoreactive processes, suggesting that theymay represent separate populations.

Our results also reveal the presence of CR-ir popula-tions in superficial tectal layers, but not in the stratummarginale (except for a few displaced perikarya in therostral tectal region). The distribution of CR-ir cells in thezebrafish tectum appears more extensive than that re-ported for the tench, another cyprinid, in which no CR-ircells were observed in the SFGS, SO, or SM (Arevalo et al.,1995). In addition, the distribution observed is more ex-tensive than that reported for gray mullet (Dıaz-Regueiraand Anadon, 2000). The finding of CR-ir fibers in the outerand inner parts of the stratum opticum is in agreementwith the expression of CR in retinal ganglion cells andfibers. A recent study in zebrafish has shown that, aftercrushing of the optic nerve, the CR-ir fibers of this layerdisappear in the contralateral tectum (Garcıa-Crespo andVecino, 2004). On the other hand, we report here thepresence of several populations of �-aminobutyric acid(GABA)-ergic cells in the optic tectum as well as a largenumber of GAD-ir boutons, indicating the importance ofinhibitory circuits in the zebrafish tectum. The finding ofGAD-ir boutons in the SM of the zebrafish tectum sug-gests that these are SM-ascending axons originating fromGABAergic cells located in deeper tectal layers. TheseSM-ascending axons might inhibit synaptic activity in thislayer, which in cyprinids and other teleosts receives a


Fig. 6. Sections through the cerebellum immunostained for CR(A,B,D,E) and GAD (C,F). A: CR-ir climbing fibers entering the innerpart of the molecular layer and decorating putative dendrites of Pur-kinje cells (arrows). B: Detail of climbing fibers endings (arrow) in thebasal part of the molecular layer. C: Section of the cerebellar bodyshowing small molecular layer GAD-ir neurons (“stellate cells”; ar-rowheads) and their beaded fibers directed in transverse direction(arrow). D: Section through the granular layer of the cerebellar body

showing a small group of CR-ir small neurons and CR-ir processes.E: Section of the cerebellar valvula showing a large CR-ir neuron(arrow) between the granular and the Purkinje cell layers, identifiedas a eurydendroid cell. F: Section of the cerebellar body passingtangentially below the Purkinje cell layer. The perikarya (arrows) andproximal dendrites of several eurydendroid cells appear decorated byGAD-ir boutons. Scale bars � 50 �m in A; 20 �m in B–F.

Figure 7


large number of excitatory parallel-like fibers (marginalfibers) from the torus longitudinalis (Ito and Kishida,1978; Poli et al., 1984; Ito et al., 2003). These marginalfibers form synapses with spines of conspicuous apicaldendritic trees of the pyramidal neurons (Meek, 1981).Our results also confirm recent reports of the presence ofa tectal cholinergic population (Clemente et al., 2004;Mueller et al., 2004). With regard to the tectal innervationby catecholaminergic fibers, our observations reveal theexistence of marked differences between medial and lat-eral regions, with a rather sharp boundary. This mightreflect differences in processing of visual signals comingfrom the ventral and dorsal regions of the retina, butdetermining its actual significance requires further inves-tigation.

The torus semicircularis, the main auditory and lateralline center of the teleost midbrain, is known to be homol-ogous to the mammalian inferior colliculus. The presentresults indicate the presence of rather abundant CR-irneurons in the torus semicircularis of zebrafish, as well asCR-ir processes, although they do not form well-definedlayers or differentiated cell populations as observed in thegray mullet (Dıaz-Regueira and Anadon, 2000). CR hasbeen observed in neurons of different nuclei of the torussemicircularis of gymnarchids but not in that of mormyr-ids (Friedman and Kawasaki, 1997). In mormyrids, theauditory part of the torus (nucleus medialis dorsalis) re-ceives a rich CR-ir innervation.

In the midbrain tegmentum, the largest CR-ir popula-tion corresponds to that observed in the perilemniscalnucleus of Wullimann et al. (1996), probably correspond-ing to the perilemniscular nucleus of Wullimann andNorthcutt (1988), a cerebellopetal nucleus identified ingoldfish and sunfish. The perilemniscal nucleus of ze-brafish also shows neurons immunoreactive for TRH (Dıazet al., 2002) and cholinergic cells (Mueller et al., 2004;present results), indicating the existence of different neu-

ronal subpopulations. Because no TRH-ir or cholinergicinnervation has been observed in the cerebellum, it isimprobable that in zebrafish these perilemniscal subpopu-lations were cerebellopetal. With regard to the presence ofcerebellar CR-ir climbing fibers, their origin appears to bethe inferior olive (see below), although the presence ofmossy-like fibers originating from CR-ir perilemniscalcells cannot be ruled out.

Isthmus

The present results reveal a large number of CR-ir cellsin the secondary gustatory/visceral nucleus, distributedsomewhat differently in different regions of the nucleus,with dorsocaudal and ventrorostral portions distinguish-able by differences in CR staining intensity and the dis-tribution of other complementary markers. The ventraland rostral regions of the SGN can be clearly distin-guished from the larger dorsal region by their rather richNPY-ir innervation, conspicuous GAD-ir innervation, andstrong CR immunoreactivity, indicating that the SGN ofzebrafish is not homogeneous. Moreover, our results withChAT immunocytochemistry indicate that the numerousChAT-ir neurons are concentrated in the ventrorostralpart, but they are lacking in the adjacent lateral nucleusof the valvula. It appears probable that the strongly CR-irventral region of the nucleus with cholinergic cells corre-sponds to the secondary visceral nucleus reported in gold-fish and catfish on the basis of its afferents from theprimary general visceral region and the immunoreactivityof its neurons to calcitonin gene-related protein (CGRP;Finger and Kanwal, 1992). Although the distribution ofCGRP in zebrafish is not known, this peptide and ChATappear to be codistributed in various cholinergic cellgroups of fish (Roberts et al., 1994; Molist et al., 1995;Anadon et al., 2000), and CGRP fibers from the gustatoryregion project to the inferior hypothalamic lobes of variousteleosts (Batten and Cambre, 1989), as with the cholin-ergic SGN cells of zebrafish. The expression of CR in cellsof the SGN appears to be conserved in teleosts, in that asimilar pattern was observed in the gray mullet, an ad-vanced teleost (Dıaz-Regueira and Anadon, 2000), and inthe trout (unpublished observations).

Although the lateral nucleus of the valvula (NLV) isclose to the SGN, it is clearly distinguishable by the ab-sence of CR-ir perikarya, the presence of a terminal field ofCR-ir varicose fibers, and the very scarce GAD-ir innerva-tion (present results). Our results also suggest that thepopulation of cholinergic cells located ventrorostral in thezebrafish SGN is not part of the NLV, unlike that previ-ously reported (Clemente et al., 2004; Mueller et al.,2004). In trout, the NLV and the SGN are separated moretopographically than in zebrafish, avoiding confusion be-tween them; in this species, there are cholinergic cells inthe SGN, but they are lacking in the NLV (Perez et al.,2000). Moreover, despite the fact that the vast majority ofNLV neurons gives rise to an outstanding projection to thecerebellar valvula and corpus in cyprinids and other te-leosts (Ito and Yoshimoto, 1990; Ito et al., 1997; Yang etal., 2004), ChAT-ir fibers were not observed in these cer-ebellar structures of trout (Perez et al., 2000) or zebrafish(Clemente et al., 2004; Mueller et al., 2004; present re-sults), suggesting that the rostral cholinergic subgroup ofthe SGN defined here in zebrafish is not part of the NLV.Because cholinergic fibers probably arising from ChAT-irNLV cells have been reported in the cerebellum of a cyp-

Fig. 7. Sections through the rostral rhombencephalon showingstructures immunoreactive to CR (A–G,I–M) and GAD (H). A: Trans-verse section at pretrigeminal levels showing different populations ofCR-ir reticular cells, including some large cells (solid arrows) andCR-ir neurons in the central gray. Note also conspicuous bundles ofCR-ir fibers as the medial longitudinal fascicle (mlf), the secondarygustatory tract (Sgt), and the raphe tract (open arrow). B: Sagittalsection passing through CR-ir reticular populations at octaval-trigeminal levels. Note the field of extensive dendritic arbors of retic-ular cells (asterisk). Open arrow, raphe tract. Rostral is at the right.C,D: Details of the large (thick arrow) and small CR-ir reticular cellsof the C,D area (boxed area in A). Thin arrows in C point to axons;arrowheads in D point to branching dendrites. E: Detail of the E area(boxed area in A) showing a CR-negative reticular cell perikaryon(star) covered of small CR-ir boutons (arrows). F,G: Transverse andsagittal sections, respectively, passing through the initial segment ofthe Mauthner cell axon (arrowheads) showing thick CR-ir fibers (ar-rows) coursing to and branching in the axon cap. Asterisk in F indi-cates the axon cap neuropil; star in F indicated a part of the Mauthnercell perikaryon. H: Detail of the Mauthner cell perikaryon (star)covered of coarse GAD-ir boutons (arrows). Arrowheads point toGAD-ir fibers. I: Detail of the boxed area in A as G showing CR-irneurons of the central gray (thin arrows). Open arrow, dorsal part ofthe medial longitudinal fascicle. J–M: Series of transverse sectionsthrough the ventral midline in caudorostral progression showing theraphe nucleus CR-ir cells (open arrows). The initial part of the heavilyCR-ir raphe tract is shown in L,M. Asterisk, tectobulbar tract showingmoderately CR-ir fibers; stars, midline meningeal blood vessel. Scalebars � 50 �m in A,B; 25 �m in C–F,H–M; 10 �m in G.


rinid, Phoxinus phoxinus (Ekstrom, 1987), whether theabsence of this innervation in zebrafish is due to method-ological constraints or represents an actual between-species difference has to be clarified.

In the zebrafish, the small nucleus isthmi is cholinergic(Clemente et al., 2004; Mueller et al., 2004; present re-sults), but it shows no CR immunoreactivity. No CR-irneurons were observed in the nucleus isthmi of the graymullet (Dıaz-Regueira and Anadon, 2000). As indicatedfor the ventrorostral part of the SGN, the GAD-ir inner-vation of the nucleus isthmi is highly characteristic.

In both zebrafish and gray mullet, there are CR-ir neu-rons in the interpeduncular nucleus (Dıaz-Regueira andAnadon, 2000; present results). A few CR-ir cells were alsoobserved in the habenula of both species (Dıaz-Regueiraand Anadon, 2000; Castro et al., 2006), indicating con-served CR expression. It is interesting to note the abun-dance of GABAergic processes in the interpeduncular neu-ropil, as well as the presence of some GAD-ir neurons inthis nucleus. This neuropil also receives ChAT-ir fibers(Mueller et al., 2004; present results), probably originat-ing from the band of ChAT-ir cells detected in the habe-nula (Mueller et al., 2004; Castro et al., 2006). Here, wealso noted faint ChAT immunoreactivity in the retroflexfascicle. This observation is consistent with results of tracttracing and ChAT immunohistochemical studies in trout(Yanez and Anadon, 1996; Perez et al., 2000).

Cerebellum and inferior olive

The zebrafish cerebellum contains a set of CR-ir struc-tures, which include pale-stained, large cells (euryden-droid cells) and scarce small neurons in the granularlayer. The eurydendroid cells (the teleost equivalent to thedeep cerebellar nuclei) also show intense CR expression inthe gray mullet (Dıaz-Regueira and Anadon, 2000). Al-though eurydendroid cells send dendrites to the molecularlayer, their shape, dendritic trees, and distribution arerather different from those of zebrafish Purkinje cells(Miyamura and Nakayasu, 2001). The small CR-ir cellslocated mainly in the region of the granular eminences(octavolateral region) of the zebrafish cerebellum mightcorrespond to the CR-ir brush cells consistently observedin the vestibulocerebellum of various mammals (see Dinoet al., 1999), in that no CR-ir parallel fibers were observed.However, further studies are necessary to investigate thispossibility. The zebrafish cerebellum receives rich CR-irinnervation by fibers ending in the deep molecular layeras characteristic small, claw-shaped bunches that are sim-ilar to the climbing fibers anterogradely labeled withhorseradish peroxidase in catfish from the inferior cere-bellar peduncle, which presumably originated from thecontralateral inferior olive (Finger, 1983). Our results alsosuggest that these CR-ir fibers represent climbing fiberscontacting proximal Purkinje-cell dendritic trees, whichwould be in agreement with the conspicuous CR expres-sion observed in the inferior olive. In the gray mullet, thecerebellopetal CR-ir fibers were located mainly at the levelof the Purkinje cells, and the inferior olive also expressedCR (Dıaz-Regueira and Anadon, 2000), which is consistentwith this hypothesis. Thus, CR appears to be a marker forclimbing fibers in some teleosts. In most mammals, sub-sets of climbing fibers also express CR (see Dino et al.,1999).

No colocalization of CR with any of the other markersstudied was observed in the zebrafish cerebellum. Another

interesting finding was the parallel fiber-like distributionof stellate cell GAD-ir axons in the molecular layer and thedistribution of GAD-ir boutons in the granular layer alongthin negative lines representing mossy fibers. This distri-bution is very different from the scattered molecular-layerGABA-ir boutons and the crown-like organization of syn-aptic boutons of Golgi cells around large glomeruli ob-served in the dogfish cerebellum (Alvarez-Otero et al.,1995). In addition, the parallel fiber-like organization ofGAD-ir stellate cell axons found in the molecular layer ofthe zebrafish cerebellum was also observed in the cerebel-lar crest over the octavolateral region.

Brainstem motor nuclei and spinalmotoneurons

The use of ChAT immunocytochemistry allowed charac-terization in detail of the distribution of these nuclei in theadult zebrafish (Clemente et al., 2004; Mueller et al., 2004;present results). The expression of CR in motor nuclei ofthe zebrafish showed great similarity to that seen in thegray mullet, suggesting that its expression pattern ishighly conserved. Specifically, visceromotor neurons lackCR in both zebrafish and mullet (Dıaz-Regueira and Ana-don, 2000), and spinal motoneurons and at least someneurons of oculomotor nuclei exhibit moderate to intenseCR immunoreactivity in both species. In zebrafish, thetrunk muscles and some extraocular muscles (the rostralrectus muscle) show strongly CR-ir motor fibers (unpub-lished observations), which is in agreement with the ob-servation of CR immunoreactivity in some oculomotoneu-ron perikarya. Furthermore, the innervation of theoculomotor nucleus by CR-ir fibers is strikingly similar inthe zebrafish and gray mullet: very thick CR-ir fibersascending in the medial longitudinal fascicle contactround perikarya of a dorsomedial subset of oculomotornucleus with CR-ir neurons, forming conspicuous calyx-like structures. In other parts of the III nerve nucleus, theneurons are finely innervated by small CR-ir boutons orig-inating from thin fibers. Heterogeneity of synapses ondifferent parts of the oculomotor nucleus has been ob-served, with reduced silver staining in the trout (Schuster,1971). We suggest that the thick CR-ir fibers may inner-vate the rostral rectus muscle subnucleus, which would besimilar to the innervation of this subnucleus in the larvallamprey by internuclear fibers forming calyces on mo-toneurons (Gonzalez et al., 1998). The origin of these thickCR-ir fibers in zebrafish might be either abducens inter-neurons or large vestibular nucleus neurons showing CRimmunoreactivity (present results), insofar as, in the gold-fish, these neurons project to the oculomotor nucleus(Torres et al., 1992). Our results in zebrafish indicate thatfast calcium-binding proteins participate in the regulationof vestibuloocular circuits involved in the horizontal nys-tagmus, which would be in agreement with the functionalspecialization revealed by physiological studies in goldfish(Pastor et al., 1991).

Motoneurons were the most conspicuous CR-ir spinalpopulation. In this regard, our results in zebrafish aresimilar to those obtained in the mullet (Dıaz-Regueira andAnadon, 2000). This appears to be an ancient character-istic of motoneurons, because both amphioxus (Castro etal., 2004) and lamprey (Megıas et al., 2003) showed CR-irspinal motoneurons. However, rat spinal motoneuronslack CR immunoreactivity (Resibois and Rogers, 1992;Ren et al., 1993).


Fig. 8. A–F: Transverse (A–C) and sagittal (D–F) sections throughthe octavolateral area (A–F) showing CR-ir neurons and fibers. A: Sec-tion showing CR-ir neurons in the magnocellular octaval nucleus(arrowhead) and dorsal (thin arrows) and ventral (thick arrow) partsof the tangential octaval nucleus. Open arrows indicate CR-ir fibers inthe octaval nerve; asterisk, lateral line region; star, field of CR-irfibers. Medial is at the left. B: Section at the entrance of the octavalnerve showing CR-ir neurons (arrows) in dorsal and ventral parts ofthe tangential nucleus. Star, field of CR-ir fibers. Medial is at the left.C: Section at the level of entrance of the glossopharyngeal nerveshowing CR-ir neurons (arrows) in the descending octaval nucleus.Asterisk, field of descending primary octaval fibers; star, field of CR-ir

fibers. Medial is at the left. The approximate location of A–C isdepicted in Figure 1H–J. D–F: Sections passing through fields ofoctaval fibers in the (stars in D,F) and terminals (asterisk in D). E isa detail of the field of octaval terminals of the boxed area in D(descending octaval nucleus). The arrows in F point to CR-ir neuronsin the descending octaval nucleus. The insets represent the approxi-mate plane of sections in a dorsal view of the brain (upper) and thelocation of the descending octaval nucleus in the section (lower).G: Section of the caudal octaval ganglion showing CR-ir bipolar neu-rons. Scale bars � 50 �m in A–D,F; 25 �m in E,G; 250 �m in lowerinset.


Figure 9


Reticular formation

The reticulospinal system of the larval and adult ze-brafish has been extensively studied with tract-tracingmethods (Kimmel et al., 1982, 1985; Metcalfe et al., 1986;Lee and Eaton, 1991). Up to 27 different types of reticu-lospinal cell have been identified in adults, three of them(the Mauthner cell and their serial homologues MiD2cmand MiD3cm) as individual pairs. Our results suggest thatmost reticulospinal cells, but not the Mauthner cells, wereCR-ir, because the medial longitudinal fascicle consistsmostly of thick CR-ir axons coursing along the medullaand spinal cord in its dorsal and ventral compartments.The number of thick CR-ir axons of the medial longitudi-nal fascicle (about 70–75 on each side of about 80–85thick axons), although less than the maximal number ofreticulospinal neurons calculated on the basis of the sumof the maximal number of cells labeled in different tract-tracing experiments (about 130–140; see Lee et al., 1993a;O’Malley et al., 2003), is not far from this figure if we takeinto consideration that the axons of a part of the reticu-lospinal neurons do not course in the medial longitudinalfascicle (a part of the rostral group and many caudalreticular cells). In addition, other reticular populationsnot previously reported to be reticulospinal showed CRimmunoreactivity. These results are similar to those ob-tained in the gray mullet (Dıaz-Regueira and Anadon,2000), which shows various CR-ir reticular groups in sim-ilar locations.

The calcium responses of reticulospinal cells of larvalzebrafish to escape-eliciting stimuli have been studied vianeuroimaging techniques (O’Malley et al., 1996, 2003;Gahtan et al., 2002). Possible roles of CR in CR-ir reticular

cells might be to modulate calcium oscillations duringactivity, although this has not been explored in this ex-perimental system. Recent studies with knockout mice inthe cerebellum indicate that the calcium-binding proteinsCR and parvalbumin have evolved as functionally dis-tinct, physiologically relevant modulators of intracellularcalcium transients (Schwaller et al., 2002).

The observation of a characteristic mat of CR-ir termi-nals, possibly originated from octaval cells, in the caparound the initial segment of the Mauthner axon is inter-esting, along with that of large GAD-ir boutons practicallycovering the Mauthner cell perikaryon and lateral den-drite but lacking on the axon cap, which is similar to thecase reported for the goldfish (Lee et al., 1993b). Compar-ison of the distribution of CR and GAD terminals on theMauthner cell suggests that the CR-ir fibers are notGABAergic, probably representing excitatory fibers. Asregards the Mauthner axon, our results in zebrafish indi-cate the absence of CR immunoreactivity, which is atvariance with that reported in another cyprinid, the tench(Crespo et al., 1998). In mormyriformes and gray mullet,the Mauthner axon is also CR negative (Friedman andKawasaki, 1997; Dıaz-Regueira and Anadon, 2000).

Octavolateral region

The octaval region of zebrafish shows CR-ir neuronsmainly in the magnocellular and the dorsal and ventraltangential octaval nuclei, first described in the cyprinidLeuciscus (Tello, 1909). CR-ir neurons have been observedin some octaval nuclei of mormyriformes (Friedman andKawasaki, 1997) and in the magnocellular nucleus but notin the tangential nucleus of mullet (Dıaz-Regueira andAnadon, 2000). These differences probably reflect differ-ent specializations of the octaval circuitry in differentteleosts. For the zebrafish octavolateral region, we haveobserved only faint CR-ir cells in the medial nucleus,whereas CR-ir structures are lacking in the cerebellarcrest, which is roughly similar to observations in graymullet (Dıaz-Regueira and Anadon, 2000). Although thelateral line region of mormyriformes exhibits CR-ir cellsand fibers (Friedman and Kawasaki, 1997), in these spe-cies it has evolved secondarily in a highly complex elec-trosensory region showing wide between-species differ-ences in CR immunoreactivity. Accordingly, comparisonwith that of nonelectroreceptive teleosts such as the ze-brafish and gray mullet is not possible.

Viscerosensory region and area postrema

The viscerosensory lobes of the zebrafish contain nu-merous CR-ir neurons and processes, both in the cortexand in more central locations. This suggests that thiscalcium-binding protein is involved in taste processing,probably influencing temporal characteristics of neuronalfiring. Our results also show extensive GAD-ir innervationof the lobes, probably originated from the small GAD-irneurons of the cortex of the lobes. In goldfish GABAergicterminals of the vagal lobe appear to be involved in mod-ulation of synapses of gustatory primary afferents (Sharpand Finger, 2002), and this might also apply to zebrafish.Unlike the goldfish vagal lobes, which show a layeredorganization of GABAergic terminals (Sharp and Finger,2002), those of the zebrafish show a rather homogeneousdistribution, probably indicating differences betweenthese species in degree of specialization for taste informa-tion processing. On the other hand, the distribution of

Fig. 9. Transverse sections through the medulla oblongata (A–I)and rostral spinal cord (J,K) showing distribution of CR (A,D,E,H,J,K), GAD (B,I), neuropeptide Y (C,G), and TH (F) immunoreactiv-ities in different areas. A: Section through the vagal lobe showingnumerous CR-ir cells in superficial (star) and central (asterisk) re-gions. Medial is at the left. B: Section through the vagal lobe showingthe rich and homogeneous GAD-ir innervation of the central neuropil(asterisk). Star, superficial region. Medial is at the left. C: Sectionthrough the vagal lobe showing a band of NPY-ir fibers (solid arrows)below the cortex (star) and also more scattered fibers (open arrow)medial to the central region (asterisk). Medial is at the left. Theapproximate location of A–C is depicted in Figure 1L. D: Detail ofCR-ir perikarya and processes in the vagal lobe. E: Transverse sectionof the area postrema showing small CR-ir cells (arrow) below theneurovascular region (arrowhead). F: Similar section through thearea postrema showing tyrosine hydroxylase-ir neurons (arrow) closeto the meningeal capillary network (arrowhead). G: Section of thearea postrema showing NPY-ir fibers. The arrowhead points to thecapillary network. The approximate location of E–G is depicted inFigure 1O. H: Section through the inferior olive (open arrows) show-ing numerous CR-ir cells. The arrowhead points to a bundle of olivo-cerebellar fibers coursing toward the midline. The solid arrow pointsto a ventrolateral bundle (octaval?) with thick CR-ir fibers. The ap-proximate location of the figure is depicted in Figure 1M. I: Section ofthe dorsal part of the inferior olive showing a GAD-ir neuron (arrow-head) and GAD-ir boutons. The arrows point to GAD-negative olivaryneurons. J,K: Sections through the ventral part of the spinal cordshowing CR-ir motoneurons (arrows) and thick CR-ir fibers of themedial longitudinal fascicle. In K, the axon initial segment of a largeCR-ir motoneuron is indicated (arrowhead). Note also the thick motoraxons. The asterisks indicate the CR-negative Mauthner axons. Starin K indicates the central canal. The approximate location of J isdepicted in Figure 1P. Scale bars � 50 �m in A–C,E–H,J,K; 25 �m inD; 12.5 �m in I.


NPY in the vagal lobes in zebrafish is rather similar tothat observed in the goldfish (Farrell et al., 2002), with avery richly NPY-ir capsular region, a sparse central zoneand a richer deep region, although the layering is not socomplex. A similar layered distribution has also been ob-served for processes of the superficial TRH-ir cells of thevagal lobe of zebrafish (Dıaz et al., 2002). The commis-sural nucleus of Cajal shows a neurochemical organiza-tion similar to that of the vagal lobe. On the other hand,the presence of some NPY-ir neurons associated with thevisceromotor column has also been reported in trout (Cas-tro et al., 1999).

Just caudal to the commissural nucleus, the zebrafisharea postrema can be distinguished by its TH-ir neuronsclosely associated with a highly vascularized meningealdorsal region (Ma, 1997; Kaslin and Panula, 2001; presentresults), as reported in the goldfish (Morita and Finger,1987). Our results indicate that a number of the blood-vessel-associated neurons contain CR and that they ex-tend rostral processes to the commissural nucleus, as alsoreported for TH-ir cells. GAD and NPY immunocytochem-istry results indicate that this region is different from thecommissural nucleus in that it practically lacks bothNPY-ir and GAD-ir fibers; this might be related to itsfunction as a primary chemosensory vascular organ(Morita and Finger, 1987).

FUNCTIONAL CONSIDERATIONS

The finding of CR in a number of cell types of thezebrafish brainstem suggests that it is involved in manyneural circuits, although it tells us little about the possiblefunctional roles of CR. It is generally considered that CR,like other closely related calcium-binding proteins (calbi-ndin 28K, parvalbumin), buffers the intracellular calciumconcentration and hence contribute to membrane proper-ties of neurons participating in the regulation of impor-tant processes, such as calcium transients, neurotrans-mitter release, and synaptic modifications (Miller, 1991;Lledo et al., 1992; Andressen et al., 1993; Arai et al., 1993;Chard et al., 1993). Recent studies in cerebellum of micelacking CR or calbindin reveal altered firing patterns ofgranule cells (Gall et al., 2003; Cheron et al., 2004): CR-deficient granule cells exhibit faster action potentials andgenerate repetitive spike discharge, results revealing thatcalcium-binding proteins modulate neuronal excitabilityand activity of cerebellar circuits. However, why some celltypes express CR, calbindin 28K, or parvalbumin and therelationship to the types of neurotransmitter used bythese cells are not clear. As regards CR expression in thevarious cells of the zebrafish brainstem, at least some cellswere either motoneurons or pertain to putative excitatorypathways (octaval primary and secondary cells, cells of thevagal and facial lobes, eurydendroid cells, secondary gus-tatory nucleus, inferior olive, many reticulospinal cells).Some of these CR-ir cells (motoneurons, some SGN neu-rons) are cholinergic, but there are cholinergic cells (forinstance, isthmic nucleus cells) that do not express CR.With regard to other complementary markers, the distri-bution of CR-ir terminals is very different from thosecontaining the GABA-synthesizing enzyme GAD in thedifferent regions and nuclei examined, suggesting that CRis not expressed by GABAergic cells. It appears probablethat most noncholinergic CR-ir cells use glutamate or

other excitatory transmitters, a question that should beaddressed in further studies.

Finally, cells of different nuclei show differences instaining intensity with CR immunocytochemistry, whichprobably reflects differences in concentration of cytoplas-mic CR among nuclei, and even among cells of the samenucleus. Although the absence of CR in the cerebellumresults in impaired motor coordination and in markedabnormalities in the Purkinje cell firing (Schiffmann etal., 1999) and CR gene inactivation in mice abolishedlong-term potentiation induction in the dentate gyrus(Schurmans et al., 1997), the possible physiological signif-icance of differences in concentration of CR between cellsis not known. With regard to proposed protective effects ofCR and other calcium-binding proteins, some studies havefound that neurodegenerative and morphogenic effects ofintrahippocampal KA injection in mice are not altered inthe absence of parvalbumin, either alone or in combina-tion with calbindin or CR (Bouilleret et al., 2000;Schwaller et al., 2002), indicating that the effects of CRlevels on the neuronal physiology are subtle.

CONCLUSIONS

Analysis of CR distribution in the zebrafish brainstemreveals a number of different cell types in the variousnerve centers, indicating its usefulness for studying thecell composition of these centers in this species. Moreover,the CR expression pattern in zebrafish shows a largenumber of features shared with an advanced teleost, thegray mullet, which indicates that the involvement of CR infunctional circuits of the teleost brain is probably ratherconservative. Together, our results indicate that CR is amarker of discrete cell populations appearing to be wellconserved throughout teleosts that may be of value as acell signature for deciphering complex brain morphoge-netic processes in the teleost group.

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Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: Distribution and comparison...

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Transcript of Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: Distribution and comparison...