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

Calretinin Immunoreactivity in the Brainof the Zebrash, Danio rerio:

Distribution and Comparison with SomeNeuropeptides and Neurotransmitter-Synthesizing Enzymes. I. Olfactory

Organ and Forebrain

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 forebrain and the olfactory system of the adult

zebrash was studied by using immunocytochemical techniques. Previous studies in troutforebrain have indicated that CR-immunoreactive neurons acquire this phenotype ratherearly in development (Castro et al., J. Comp. Neurol. 467:254269, 2003). Thus, preciseknowledge of CR-expressing neuronal populations in adult zebrash may help to decipherlate stages of forebrain morphogenesis. For analysis of some forebrain nuclei and regions, CRdistribution was compared with that of various ancillary markers: choline acetyltransferase,glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y, thyrotropin-releasinghormone, and galanin. The results reveal that calretinin is a specic marker of olfactoryreceptor neurons and of various neuronal populations distributed throughout the telenceph-alon and diencephalon. In addition, CR immunocytochemistry revealed characteristic pat-terns of bers and neuropil in several telencephalic and diencephalic regions, indicating thatit is a useful marker for characterizing a number of neural centers, pathways, and neuronalsubpopulations in the zebrash forebrain. Some ancillary markers also showed a distinctivedistribution in pallial and subpallial regions, revealing additional aspects of forebrain orga-nization. Comparison of the distribution of CR observed in the forebrain of zebrash with thatreported in other teleosts revealed a number of similarities and also some interestingdifferences. This indicates that various neuronal populations have maintained the CR phe-notype in widely divergent teleost lines and suggests that CR studies may prove very usefulfor comparative analysis. J. Comp. Neurol. 494:435459, 2006. 2005 Wiley-Liss, Inc.

Indexing terms: calcium-binding proteins; immunohistochemistry; glutamic acid decarboxylase;neuropeptide Y; forebrain; zebrash; Danio rerio

Calretinin (CR) is a 29-kD calcium-binding protein ofthe EF-hand family, which was rst identied by genecloning from chick retina (Rogers, 1987). The EF-handcalcium-binding proteins have been employed as selectiveneuronal markers, because they are expressed principallyby distinct groups of neurons in both the central andperipheral nervous system (Celio, 1990; Baimbridge et al.,1992; Resibois and Rogers, 1992; Andressen et al., 1993).Major populations of calretinin-immunoreactive (CR-ir)cells have been identied in various regions of the ratforebrain and hindbrain (Arai et al., 1991; Jacobowitz and

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 17 June 2005; Accepted 26 July 2005DOI 10.1002/cne.20782Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 494:435459 (2006)

2005 WILEY-LISS, INC.

Winsky, 1991). Calcium-binding proteins have been sug-gested to contribute to calcium homeostasis by bufferingthe intracellular free calcium concentration (Miller, 1991;Baimbridge et al., 1992; Lledo et al., 1992; Chard et al.,1993). Both CR and calbindin 28K (CB) may protect neu-rons against calcium increases during periods of high-frequency discharge or neurotransmitter-induced depolar-ization or in pathological conditions such as ischemia orepileptic seizures (Andressen et al., 1993; Freund andMagloczky, 1993; Brion and Resibois, 1994). Furthermore,CR- and CB-ir neurons in the cerebral cortex are resistantto degenerative processes in Alzheimers disease (Hof andMorrison, 1991; Hof et al., 1993).

The zebrash is a model vertebrate for developmentalresearch, and numerous studies have dealt with braindevelopment in this species. However, most of these stud-ies have been limited to the early embryonic period, andearly neuronal populations have generally not been fol-lowed beyond 48 or 72 hours post fertilization. Hence,there is a signicant gap between our knowledge of ze-brash brain organization at early stages and in the adult.Some recent studies have attempted to ll this gap bystudying neurochemical organization in the adult brain(Kaslin and Panula, 2001; Rink and Wullimann, 2001,2002; Daz et al., 2002; Ma, 2003; Clemente et al., 2004;Kaslin et al., 2004; Mueller et al., 2004). A detailed knowl-edge of the adult brain is important for understandingchanges caused in the brain by experimental manipula-tion, mutation, or genetic approaches.

A previous study in an advanced teleost has shown theusefulness of CR immunocytochemistry for characterizinga wide variety of neuronal populations throughout thebrain (Daz-Regueira and Anadon, 2000). A recent studyin the trout telencephalon has also shown that CR immu-noreactivity is expressed early in specic neuronal popu-lations, which maintain this expression throughout devel-opment and adulthood (Castro et al., 2003). Thus,knowledge of these populations may help in the compari-son of the developing and adult brains. As far as we areaware, the distribution of CR has not previously beenstudied in either the developing or adult brain of ze-brash.

The aim of the present study (and of a forthcomingpaper, Castro et al., 2006) was to characterize the cellpopulations and tracts immunoreactive to CR in the adultzebrash brain. A further aim was to compare the distri-butions of CR and other markers (glutamic acid decarbox-ylase, tyrosine hydroxylase, choline acetyltransferase,neuropeptide Y, thyrotropin-releasing hormone, and gala-nin), which have been shown to be useful for characteriz-ing regions and nuclei in the trout telencephalon (Castroet al., 2003).

MATERIALS AND METHODSTwenty-seven adult zebrash (Danio rerio, Cyprinidae)

were obtained from a local supplier and maintained ap-propriately in an aquarium. Prior to the experiments the

Abbreviations

AC anterior commissuresAPC accessory pretectal nucleusAT anterior thalamic nucleusATN anterior tuberal nucleusCIL central nucleus of the inferior lobeCP central posterior thalamic nucleusCSP commissure of the supercial pretectal nucleiDAO dorsal accessory optic nucleusDc central zone of the dorsal telencephalic areaDd dorsal zone of the dorsal telencephalic areaDl lateral zone of the dorsal telencephalic areaDla anterior region of the DlDld dorsal region of the DlDlv ventral region of the DlDm medial zone of the dorsal telencephalic areaDma anterior region of the DmDp posterior zone of the dorsal telencephalic areaDP dorsal posterior thalamic nucleusDTN dorsal tegmental nucleusEcl external cellular layerEn entopeduncular nucleusfr fasciculus retroexusGl glomerular layer of the olfactory bulbH habenulaHC horizontal commissureHYc caudal hypothalamusHYv ventral hypothalamusIII oculomotor nerveIP interpeduncular nucleuslfb lateral forebrain bundleLL lateral lemniscusLR lateral recessLRN lateral recess nucleusMB mammillary bodymfb medial forebrain bundlemlf medial longitudinal fascicleMPo magnocellular preoptic nucleusNDLI diffuse nucleus of the inferior hypothalamic lobeNLV nucleus lateralis valvulae

Nmlf nucleus of the medial longitudinal fascicleOB olfactory bulbOC optic chiasmOT optic tectumOtr optic tractOVLT vascular organ of the terminal laminaP pineal organPa parapineal organPC posterior commissurePGa anterior preglomerular nucleusPGl lateral preglomerular nucleusPGm medial preglomerular nucleusPHL posterior hypothalamic lobePL perilemniscal nucleusPpa anterior part of the parvocellular preoptic nucleusPpp posterior part of the parvocellular preoptic nucleusPSM magnocellular part of the pretectal supercial nucleusPSP parvocellular part of the pretectal supercial nucleusPTN posterior tuberal nucleusPTO pretoral nucleusRC commissure of the raphe tractSQ suprachiasmatic nucleusSG subglomerular nucleusTGN tertiary gustatory nucleusTL torus longitudinalisTLA torus lateralisTP periventricular nucleus of posterior tubercleTPM pretectomammillary tract and nucleusTS torus semicircularisVC cerebellar valvulaVc central nucleus of the ventral telencephalic areaVd dorsal nucleus of the ventral telencephalic areaVL ventrolateral formationVl lateral nucleus of the ventral telencephalic areaVM ventromedial thalamic nucleusVp posterior nucleus of the ventral telencephalic areaVs supracommissural nucleus of the ventral telencephalic

areaVv ventral nucleus of the ventral telencephalic area

436 A. CASTRO ET AL.

specimens were anesthetized with 0.1% 3-aminobenzoicacid ethyl ester methane sulfonate salt (MS-222; Sigma,St. Louis, MO) in freshwater. All experiments conformedto the European Communitys guidelines on animal careand experimentation.

Western blottingBrains of seven adult zebrash were mechanically ho-

mogenized in 200 l of lysis buffer containing 50 mMTris (pH 7.6), 5 mM EDTA, 150 mM NaCl, 2 M2-mercaptoethanol, 4 mM leupeptin, 0.3 M aprotinin, 4mM pepstatin, and 0.1 mM phenylmethylsulfonyl uoride(PMSF) and were then centrifuged. Proteins and peptidesin the supernatant were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on15% acrylamide 80 50 1-mm slab gels, at a constant100 V for 1.5 hour (Mini-Protean II PAGE system, Bio-Rad, Richmond, CA). Then 2025 g of protein in 2Xsample buffer (100 mM Tris-HCl, pH 6.8, 1% SDS, 4%2-mercaptoethanol, 0.02% brilliant blue G, and 24% glyc-erol) were applied to each lane, and molecular weightsstandards (SigmaMarkers; Sigma) were run in additionallanes. The separated proteins were then electroblotted ina Transblot SD system (Bio-Rad) onto a nitrocellulosemembrane (0.2-m pore size; Sigma) at 100 V for 1 hour.

Nonspecic binding sites were blocked by incubationovernight in 5% milk powder in 0.01 M phosphate-buffered saline (PBS; pH 7.4) containing 0.2 % Tween-20(PBS-Tween) at 4C. The blots were then incubated for 20hours at room temperature in a 1:1,000 dilution of theanti-CR antiserum (Swant, Bellinzona, Switzerland) inPBS-Tween. This antiserum was raised against puriedrecombinant human CR (for the sequence of human CR,see Parmentier and Lefort, 1991). After three rinses inPBS, the blots were incubated in goat anti-rabbit IgG(Dako, Glostrup, Denmark; diluted 1:100 in PBS-Tween)for 1 hour at room temperature and then rinsed in PBSand incubated in peroxidase-antiperoxidase complex(PAP; Dako) diluted 1:200 in PBS-Tween for 1 hour atroom temperature. Staining was visualized by the diami-nobenzidine (DAB) reaction as described below, intensi-ed with 0.03% cobalt chloride. For comparative purposes,brain extracts of two trouts and two rats were subjected toan identical analysis.

Calretinin immunohistochemistryFor CR immunohistochemistry, zebrash were xed by

immersion or by transcardial perfusion with 4% parafor-maldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Theirbrains were then dissected out and left in xative for 24hours. Most specimens were cryoprotected in 30% sucrosein PB, frozen with liquid nitrogen, and cut on a cryostat.The whole heads or brains were cut in transverse or sag-ittal planes at a thickness of 1225 m on a cryostat,mounted on gelatinized slides, and processed for immuno-cytochemistry. Two paraformaldehyde-xed adult brainswere embedded in 3% agarose in PB and cut on a Vi-bratome (50100 m in thickness).

Sections (either cryostat or Vibratome sections) wereprocessed for CR immunocytochemistry as follows: 1) sev-eral rinses in 0.01 M PBS, pH 7.4; 2) treatment with 3%H2O2 in PBS for 30 minutes to block endogenous peroxi-dase activity; 3) two rinses in PBS (10 minutes each); 4)treatment with normal goat serum (Dako, Glostrup, Den-mark) diluted 1:10 in PBS for 1 hour; 5) incubation with

rabbit CR antiserum (SWant; dilution 1:1,000 in PBS) for2072 hours at 4C in a moist chamber; 6) two rinses inPBS (10 minutes each); 7) incubation with goat anti-rabbitimmunoglobulin (Dako; diluted 1:50 in PBS) for 1 hour; 8)two rinses in PBS (10 minutes each); 9) incubation withperoxidase-antiperoxidase complex (PAP; Dako; diluted1:200 in PBS) for 1 hour; 10) two rinses in PBS (10 min-utes each); and 11) two rinses in Tris-HCl buffer, pH 7.6(10 minutes each). Finally, the immune complex was vi-sualized by incubation with 0.06% 3,3-DAB (Sigma) and0.005% H2O2 in Tris-HCl buffer for 510 minutes. Thesections were then rinsed in Tris-HCl buffer, dehydrated,and coverslipped. Free-oating Vibratome sections wereprocessed as above, although the time of incubation ineach antiserum was doubled; nally, they were mountedon slides and dried at room temperature for 20 hoursbefore coverslipping. The specicity of the immunostain-ing was tested by replacement of the primary antiserumwith normal goat serum alone and by preabsorption of theprimary antiserum, diluted 1:1,000 in PBS, with recombi-nant human CR (SWant, 0.172 M). Following both ofthese controls, no immunoreactivity was observed in anybrain region.

Other methodsAs ancillary markers for characterizing forebrain re-

gions better, we also used series of transverse sectionsimmunostained with antibodies to neuropeptide Y (NPY;Sigma, dilution 1:1,000), glutamic acid decarboxylase(GAD; Chemicon, Temecula, CA, dilution 1:1,000),thyrotropin-releasing hormone (TRH; Biogenesis, Poole,Dorset, UK, dilution 1:2,000), tyrosine hydroxylase (TH;Chemicon, dilution 1:1,000), choline acetyltransferase(ChAT; Chemicon, dilution 1:100), and galanin (Gal; Af-nity, Mamhead, UK, dilution 1:2,000). The protocols andcontrols used were as published elsewhere for the ze-brash (Daz et al., 2002), trout (Castro et al., 1999; Perezet al., 2000; Daz et al., 2001; Rodrguez et al., 2003;Anadon et al., 2002), and dogsh (Sueiro et al., 2004). Inaddition, we used Nissl-stained series of the adult ze-brash brain.

Photomicrographs were taken with a digital camera(DP12, Olympus, Tokyo, Japan), converted to gray scale,and adjusted for contrast and brightness with Corel Pho-toshop (Corel, Ottawa, Canada).

The nomenclature used in this study is based on that ofthe zebrash brain atlas of Wullimann et al. (1996).

RESULTSThe CR antibody used in this study is well characterized

and has been tested by Western blotting in brain extractsof teleosts and other actinopterygians (Daz-Regueira andAnadon, 2000; Huesa et al., 2002; Castro et al., 2003).Here we investigated by Western blotting whether theantibody recognized a protein of the appropriate molecu-lar weight in the zebrash brain. Western blotting ofprotein extracts of zebrash brain reveals a single band ofabout 2829 kD, an MW slightly lower than those re-vealed in trout and rat brain extracts (29 kD; Fig. 1).Incubation of the sections either with the primary anti-body preadsorbed with CR or without the primary anti-body produced no staining of these structures, indicatingthat the staining is specic for CR.

437CALRETININ IN THE ZEBRAFISH FOREBRAIN

Immunocytochemistry with the anti-CR antibody re-vealed neurons and bers distributed in the brain (Figs. 2,3), as well as olfactory neurons (Fig. 2). Figure 3 showsschematically the distribution of CR-immunoreactivestructures observed in transverse sections of the zebrashforebrain. In general, the distribution observed in thebrain has many similarities with that observed in the greymullet (Daz-Regueira and Anadon, 2000), but there are anumber of differences.

Olfactory systemIn the olfactory organ (olfactory rosette), numerous

small CR-ir bipolar cells were observed in the neuroepi-thelium of the olfactory lamellae (Fig. 2A). These cells hadperikarya located throughout the height of the epithelium,as well as rather thin apical dendrites reaching the olfac-tory organ surface (Fig. 2B). High magnication of theapical surface showed the presence of CR-ir cilia in thedistal knob of receptor cells (ciliated receptor neurons),which probably make up the majority of the CR-ir cellsobserved (Fig. 2B,C), although the presence of CR-ir mi-crovillar receptor cells could not be ruled out. No CR-ircrypt cells were observed. A number of CR-ir bers origi-nating from the olfactory epithelium coursed in smallbundles to meet in a thick olfactory nerve just below therosette. The olfactory nerve was intensely CR-ir (Fig. 2A),coursing to the olfactory bulb.

In the olfactory bulbs, most CR-ir structures observedwere terminal elds of olfactory bers in the olfactoryglomeruli and plexus (Fig. 2DH). Different glomerularelds could be distinguished by the appearance and inten-sity of staining of the olfactory terminals: on the basis oftheir relative location in the bulb, we term these CR-irelds the rostral, dorsolateral, dorsomedial, ventrolateral,

and ventromedial elds. Although our ventromedial eldroughly corresponds to the ventral group of Baier andKorsching (1994), an unambiguous correspondence cannotbe determined because of the different section plane andmethodology used here and because CR is not expressed inall glomeruli. Likewise, our dorsolateral eld probablycorresponds in part to the dorsal group of Baier and Kor-sching (1994), our dorsomedial eld to the medial group,and our ventrolateral eld to the lateral group of theseauthors. The most prominent CR-ir glomeruli were thoseof the rostral and dorsolateral elds (Fig. 2D,E), whereasin the ventrolateral eld most of the glomeruli were smalland inconspicuous (Fig. 2G,H). In the ventromedial eld,those glomeruli located dorsomedially were rather prom-inent (Fig. 2F).

Whereas most glomeruli of the rostral, dorsolateral,ventrolateral, and ventromedial elds exhibited strong CRimmunoreactivity, the large glomeruli of the dorsomedialeld (dorsal glomeruli of Baier and Korsching, 1994) ex-hibited only faintly CR-ir bers. Glomerular elds arerevealed by TH immunocytochemistry, because they re-ceive a large number of processes of TH-ir periglomerularcells; they are also revealed by rich GAD-ir innervation.CR-ir bers were very scarce or absent in the inner cellu-lar layer of the olfactory bulbs, and no bulbar CR-irperikarya were observed.

Telencephalon and preoptic regionCalretinin. The telencephalic hemispheres of teleosts

are generally subdivided into a dorsal telencephalic area(pallium) and a ventral telencephalic area (subpallium;see Northcutt and Braford, 1980; Wullimann and Rink,2002). In adult zebrash, both these areas contained CR-irperikarya and bers showing a characteristic distribution,

Fig. 1. Western blots of brain extracts stained with the rabbit polyclonal anti-calretinin antibodyshowing a single band of about 2829 kD in the zebrash lane (Z). The two other bands correspond totrout (T) and rat (R) brain extracts run in parallel. Molecular weight markers (kD) are at the left.


Fig. 2. Photomicrographs showing calretinin immunoreactivity inthe olfactory organ (AC) and olfactory bulb (DH). A: Low-magni-cation view of olfactory lamellae exhibiting dense areas of CR-irbipolar neurons (solid arrows). The outlined arrow points to a CR-irbundle coursing toward the olfactory nerve (ON). White stars indicatepigment cells. B: Detail of the olfactory epithelium showing CR-irbipolar neurons. The arrows point to the apical surface of neuronsexhibiting numerous cilia. Arrowhead, basal axon. C: High magni-

cation of the apical surface shows distal knobs (arrows) of bipolarneurons bearing cilia. D: Transverse section of the olfactory bulbshowing elds of CR-ir glomeruli. E,F: Detail of the boxed areas in Dshowing dorsolateral and ventromedial glomerular elds, respec-tively. G,H: Fields of small CR-ir glomeruli in the caudal ventrolat-eral region of the bulb. Note in DH the absence of CR-ir perikarya inthe olfactory bulb. Scale bars 100 m in A,D; 50 m in EH; 12.5m in B; 5 m in C.


Figure 3


Fig. 3. AO: Schematic drawings of transverse sections of thebrain of zebrash showing at the right the distribution of calretinin-immunoreactive (CR-ir) perikarya (dots) and elds rich in CR-ir pro-cesses (shaded areas). Differences in process richness are representedby gradations in the gray scale (darker richer). Some conspicuouslyCR-ir ber bundles, tracts, and commissures are also indicated bydashes or shading. The main nuclei and regions are indicated sche-matically on the left half of the sections: the nomenclature used for

brain structures was mostly based on the zebrash brain atlas byWullimann et al. (1996). The black arrows in IO point to the CR-irraphe tract decussating in the postoptic region. Crosses in BF indi-cate the telencephalic ventricles, stars in HM indicate the mesence-phalic ventricle, and asterisks in MO indicate the fourth ventricle.The location of transverse sections is indicated by lines in the gurinerepresenting the whole brain. For abbreviations, see list. Scale bar 250 m in I (applies to AI), O (applies to JO).


Fig. 4. Photographs of sections of the zebrash telencephalon,arranged in series from rostral to caudal, showing the comparativedistribution of immunoreactivity to calretinin (CR; A1A7), glutamicacid decarboxylase (GAD; B1B7), neuropeptide Y (NPY; C1C5),galanin (Gal; C6C7), tyrosine hydroxylase (TH; D1,D3D7), andthyrotropin-releasing hormone (TRH; D2). Note that forebrain re-

gions can be distinguished by large differences in the content of thesesubstances, which combined serve as a molecular signature of theregions. 1 and 2 (in A4A5, B2B6, and C1C3) indicate dorsal andventral parts of the Dm. For abbreviations, see list. Scale bars 100m in A1D7.

Fig. 5. Photomicrographs of thick (A, G-I) and thin (B-F, J-L)sections showing details of calretinin immunoreactivity in differentparts of the telencephalon and preoptic area. A: Section showing therich CR-ir innervation of the precommissural Dm. Arrows point tofascicles of afferent axons crossing the Dc. B: Detail of the CR-irinnervation of the Dm. Note that perikarya are CR negative. C: Sec-tion showing CR-ir perikarya in the Dd (large arrow) transition to theDc (double arrow) and Vs. The small arrow points to fascicles ofafferent bers. D: Detail of Dd CR-ir neurons. E: Detail of the DcCR-ir cell population. Arrow, CR-ir ber bundle. F: Section of the Dlshowing sparse CR-ir innervation. G: Section showing the CR-ir cellpopulation of yhr Vs (arrow). H: Section through the postcommissural

subpallium and anterior preoptic region showing CR-ir neurons in theVp and Ppa. The outlined arrow points to the lateral forebrain bundleand associated CR-ir cell population. I: Detail of the lateral forebrainbundle (outlined arrow) and associated cells of the dorsal entopedun-cular nucleus (solid arrow). The arrowhead points to a migrated VpCR-ir cell. J,K: Details of CR-ir cells in the Vs and Vp. Star in J,anterior commissure. L: Detail showing numerous CR-ir neurons inthe anterior parvocellular preoptic nucleus. The cells in the bottom ofthe preoptic recess (arrowhead) are in the vascular organ of theterminal lamina. For abbreviations, see list. Scale bars 100 m inA,H; 50 m in C,G,I,L; 12.5 m in B,D,E,F,J,K.


Figure 6


which allowed identication of both richly CR-ir inner-vated zones and CR-ir neuronal populations. The generaldistribution of CR-ir neurons, bers, and terminal elds inthe zebrash telencephalon is shown in Figures 3AF and4A1A7. The zebrash telencephalon has been chartedpreviously on the basis of Nissl-Bodian silver proteinate-stained series (Wullimann et al., 1996). The zones and cellgroups revealed in the dorsal area by CR immunocyto-chemistry showed some variations compared with the lim-its observed in this previous study. To characterize thesepallial zones better, we also used the ancillary markersGAD, NPY, galanin, TH, TRH (Fig. 4B1D7), and ChAT(Fig. 6F).

The dorsomedial part of the D (Dm) was very richlyinnervated by CR-ir bers, except in its rostral region(Figs. 4A1A7, 5AC,G). The Dm received a large numberof CR-ir bers in thick fascicles coursing in both the lat-eral and medial telencephalic bundles. As seen in sagittalsections, the probable origins of most of these CR-ir bersare the CR-ir cell populations of the preglomerular-mammillary nuclei (see below), but some bers might alsoarise from CR-ir pallial and subpallial cells. CR-ir berswere scarce in the most rostral region of the Dm, but morecaudally CR-ir innervation increased noticeably, althoughgradually disappearing at levels caudal to the anteriorcommissure. Moreover, the Dm-Dd region showed a con-spicuous CR-ir cell population. At precommissural levels,a few CR-ir perikarya appeared at the dorsolateral borderof the Dm, i.e., the sulcus ypsiloniformis. The number ofCR-ir neurons increased at commissural levels, althoughnone were present at postcommissural levels. This CR-irDm/Dd population occupied the dorsolateral region of thiszone, crossing the sulcus ypsiloniformis and extending tothe dorsal region (Dd) of Wullimann et al. (1996), wherethey were rather abundant (Fig. 5C,D). At caudal levels ofthe Dd, a few CR-ir cells extended ventrally from thelateral Dd, entering between the caudolateral part of thecentral region (Dc) and the medial part of the lateralregion (Dl) of the dorsal area (Fig. 5C,E).

In the rostral dorsal area, a small CR-ir cell populationwas located ventrally near the boundary with the subpal-lium or ventral telencephalic area (V; Fig. 3B), in a ventralterritory not named by Wullimann et al. (1996). In thecaudal dorsal area, a scattered population of CR-ir cells

was observed in the posterior region (Dp) extending fromlateral levels to the anterior commissure and dorsal to thedorsal entopeduncular nucleus toward medial regions.This Dp cell population did not invade the lateral region(Dl) of Wullimann et al. (1996). On the other hand, Dp, Dc,Dl, and the anterior region of D showed scarce CR-irinnervation (Figs. 3AF, 4A1A7, 5F).

Different CR-ir cell populations were distinguished inthe ventral telencephalic area. The most rostral V cellpopulations were found in the dorsal and ventral regionsof the precommissural V (Vd and Vv). In the Vv, CR-ircells were observed from just caudal to the olfactory bulbs,forming widely separated groups with scattered cells. TheVd CR-ir population was continuous with the fairly abun-dant small CR-ir cells of the supracommissural part of theV (Vs), below the richly CR-ir innervated Dm (Figs. 3DE,5G). In the Vs, some CR-ir cells also occupied ventrolat-eral locations (Fig. 5J). In sagittal sections, Vd-Vs CR-ircell populations formed a continuous oblique band. Thepostcommissural region of the V (Vp) showed pear-shapedCR-ir neurons clearly larger than those of the medial Vs(Figs. 3F, 5H,K). These Vp CR-ir cells had thick dendritesdirected laterally and branching in a neuropil at interme-diate level. The medial regions of the V contained abun-dant CR-ir bers, but this innervation was much lessdense than that of the Dm.

In the preoptic region, caudal to the anterior commis-sure, numerous CR-ir neurons were observed in the ante-rior parvocellular preoptic nucleus (Ppa), both in a medial(periventricular) row of cells and in a cell row located morelaterally extending from the ventral subpial region justlateral to the preoptic recess toward the lateral region ofthe Ppa (Figs. 3F, 5H,L). The midline CR-ir cells observedbelow the preoptic recess can be considered as part of thevascular organ of the terminal lamina (OVLT; Fig. 5L).Some cells located dorsolaterally in the posterior parvo-cellular preoptic nucleus (Ppp) had rather thick dendritesthat extended rostrally toward the postcommissural Vregion. More caudally in the preoptic region, scarce CR-irneurons were found at intermediate levels in the magno-cellular part of the preoptic nucleus (MPo; Fig. 3G).

At caudal telencephalic-preoptic levels, some CR-ir cellsof bipolar appearance were observed extending along theCR-ir ber fascicles of the lateral forebrain bundle (lfb), asseen in sagittal sections, and more caudally a small groupof CR-ir cells occupied a position dorsal to this bundle(Figs. 3F, 5I). The ventral entopeduncular nucleus com-prised a large number of small CR-negative cells, but inthe dorsal entopeduncular nucleus, some small CR-ir cellswere observed.

A large number of CR-ir bers coursed in the lateralforebrain bundle (Fig. 5H,I). At commissural and precom-missural levels, small fascicles of CR-ir bers coursedtoward the Dm crossing the Dc, seemingly giving rise tomost of the heavy CR-ir innervation of the Dm (Figs.3CE, 4A4,A5, 5A).

Other immunocytochemical markersGlutamic acid decarboxylase. The distribution of GAD

immunoreactivity in the zebrash telencephalon revealeda sharp contrast between dorsal (pallial) and ventral (sub-pallial) telencephalic regions, as well as a number of dif-ferences among divisions of the dorsal area (Figs. 4B1B7,6AD). The ventral area was very richly innervated byGAD-ir bers, whereas only faint or very faint GAD-irperikarya were seen in periventricular regions. The dorsal

Fig. 6. Photomicrographs of sections of the telencephalon showingselected structures immunoreactive to glutamic acid decarboxylase(GAD; AE), choline acetyltransferase (ChAT; F) and galanin (G,H).A: Precommissural dorsal area showing differences in GAD-ir inner-vation among regions. Note that the dorsal Dm (1) receives a denserGAD-ir innervation than the ventral Dm (2). B: Section showing thecontrast between very rich GAD-ir innervation of the Vd and the scantinnervation of the ventral Dm (2). C: Section showing differences inGAD-ir innervation between the Dp and anterior Dl. Faint GAD-irneurons (arrowheads) are appreciable in several pallial regions.D: Detail of the Dp showing GAD-ir perikarya (arrowheads) andGAD-ir boutons. E: Detail showing dense GAD-ir innervation of thevascular organ of the terminal lamina (arrow). F: Photomicrographshowing the specic innervation of the Dm by ChAT-ir bers. G: Sec-tion through the precommissural subpallium showing its richgalanin-ir innervation. H: Galanin-ir perikarya (arrowheads) are nu-merous in the anterior parvocellular preoptic nucleus. Note the ab-sence of galaninergic innervation of the vascular organ of the terminallamina (arrow). For abbreviations, see list. Scale bars 50 m inAC, FH; 25 m in D,E.


area characteristically had scattered moderate to strongGAD-ir perikarya in most divisions (Fig. 6C,D) and low,moderate, or rich GAD-ir innervation in different regions,allowing clear differentiation between some zones andsubzones (Fig. 4B1B7). In the Dm, GAD-ir innervationwas notably scant in the ventral subdivision (2 in Figs.4B2B6, 6A,B) and moderate in the dorsal division (1 inFigs. 4B2B6, 6A). Moreover, the Dc is clearly recogniz-able in the precommissural region as a division withscarcer GAD-ir bers than the Dm and Dl (Figs. 4B3B5,6A); this feature suggests that such a Dc is lacking atcommissural and postcommissural levels. The density ofGAD-ir bers was similar in the Dd and the adjacent Dldivision. The GAD-ir innervation of Dl was moderate,although some differences in density of innervation werenoted among rostral, dorsal, ventral, and caudal parts.GAD-ir innervation in the Dp was in general more richthan that of the Dl (Fig. 4B3B7), which can be used fortracing their limits.

In the ventral area, GAD-ir innervation was rather richthroughout most parts (Figs. 4B2B7, 6B) and was clearlyricher than that of the dorsal area, which here is inter-preted as a subpallial characteristic. A rich GAD-ir inner-vation was also observed in the preoptic region, both in theneuropil between periventricular cell layers and in thelateral neuropil (Fig. 4B6,B7). The vascular organ of theterminal lamina received rich GAD-ir innervation (Fig.6E).

Choline acetyltransferase. Cholinergic innervationwas very scant in most dorsal telencephalic regions, whichalso lacked ChAT-ir perikarya. However, moderate butcharacteristic ChAT-ir innervation was observed in thedorsal subdivision of the Dm, mainly at commissural lev-els (Fig. 6F). ChAT-ir bers were also observed in theventral telencephalic area coursing near the dorsal area.In the preoptic region moderately stained ChAT-ir neu-rons were found scattered along the dorsal neuropil justlateral to the anterior parvocellular and magnocellularparts of the preoptic nucleus. This preoptic neuronal pop-ulation is the most probable origin of the bers observed inthe Dm. Some magnocellular preoptic neurons also exhib-ited faint ChAT immunoreactivity.

Galanin. In the zebrash brain, Gal-ir perikarya wereobserved in four regions: the preoptic nucleus, the lateralrecess nucleus, the midbrain tegmentum, and the commis-sural nucleus of Cajal. Most Gal-ir neurons were observedin the rostral preoptic nucleus, either in ventrolateral,laterodorsal, or dorsomedial locations (Figs. 4C7, 6H). Inthe telencephalon, Gal-ir bers were mostly restricted tosubpallial regions (Figs. 4C6, 6G) and were much moreabundant in caudal than in rostral parts. In the preopticregion, Gal-ir bers were rather abundant (Fig. 6H) andran caudally as conspicuous bands of bers at the bound-ary between the hypothalamus and thalamus, richly in-nervating the posterior hypothalamic lobe. A few smallGal-ir perikarya were observed in the dorsal periventricu-lar hypothalamus, just above the lateral recess, and arather more abundant Gal-ir cell population was found inthe midbrain tegmentum medial to the lateral lemniscus.A few Gal-ir bers coursed in the brainstem to the raphe.

Neuropeptide Y. Our results reveal the existence ofseveral different subdivisions in the dorsal area showingNPY-immunoreactive bers. NPY-ir innervation was veryhigh in the dorsal part of the precommissural Dl (Dld) andalso in the rostral Dm and Dl (Figs. 4C1C4, 7A,B). These

regions contained numerous NPY-ir boutons. In contrast,NPY-ir innervation was very sparse in the Dp and sparseto moderate in the caudal Dl and rostral Dd (Fig. 4C3C5).At precommissural levels alone, NPY immunocytochemis-try clearly distinguished the Dl from the Dp, from which itwas separated by a continuous row of neurons, and in thisregion the dorsal and ventral parts of the Dl could bedifferentiated by their NPY-ir innervation (Fig. 7A).

There was also a very conspicuous NPY-ir neuronalpopulation in the lateral subpallium (Vl), as well as asmaller population in the Vd at precommissural levels.The NPY-ir cells of the Vl formed a large population ofcells located below the entorhinal sulcus at the lateralsurface and extending from the retrobulbar region to thepostcommissural region intermingled with lateral fore-brain bundles (Fig. 7C). These cells exhibited strong stain-ing and formed a continuum. Because this NPY-ir cellpopulation showed rather homogeneous characteristicsalong its length, here it is considered part of a singlenucleus. The caudal part of this population extended intothe entopeduncular nucleus, dorsal part of Wullimann etal. (1996). The NPY-ir cell population of the Vd consistedof small groups of moderately stained cells scattered in theVd at precommissural or supracommissural levels. NoNPY-ir cell population was observed in the entopeduncu-lar nucleus of zebrash (entopeduncular nucleus, ventralpart, of Wullimann et al., 1996), although some caudal Vlcells may intermingle with the most rostral portion of thisnucleus, which consists of a densely clustered mass ofmuch smaller cells, markedly unlike the organization seenin the Vl.

Thyrotropin-releasing hormone. The distribution ofTRH-ir neurons and bers in the zebrash telencephalonhas already been reported (Daz et al., 2002). Here, wemade only a determination of the rich band of TRH-irbers observed in pallial regions (Fig. 4D2; see also Fig.1BD in Daz et al., 2002). Comparison of this band withother markers indicated that it extended in the ventralsubdivision of the Dm along the precommissural region.Other pallial regions, except for the DP, showed scarceTRH-ir bers.

Tyrosine hydroxylase. Our results with TH immunocy-tochemistry revealed a number of small TH-ir neurons inthe olfactory bulb (Fig. 4D1). In addition, TH-ir cell pop-ulations were seen in the telencephalic lobes in the sub-pallium (Figs. 4D3D5, 7D,E). These populations ex-tended caudally from a retrobulbar area, ascending fromthe central nucleus of the area ventralis (Vc) through theVd, to the Vs and ending in an intermediate region be-tween the Vs and Dl that lay outside the cytoarchitectoni-cally regions dened in Wullimann et al.s study (1996), asnoted previously (Rink and Wullimann, 2001). A numberof TH-ir processes extended from this population into thecaudal region of the dorsal area here named the Dp (Figs.4D4D7, 7E,F).

In the preoptic region, we found several TH-ir cell pop-ulations. The most rostral extended from the postcommis-sural region of the preoptic recess, forming a vertical bandfrom the ventral meninges (anterior part of the parvocel-lular preoptic nucleus). More caudally, this populationextended below the preoptic recess associated with a largemeningeal blood vessel that lies in front of the optic chi-asm, forming a horizontal band here considered the vas-cular organ of the terminal lamina (Fig. 7G). The OVLTwas associated with two more lateral TH-ir preoptic pop-


ulations that continued to a more lateral position at thelevel of the optic chiasm. The OVLT gave rise to midlineTH-ir bers that extended caudally. At the level of theoptic chiasm, another TH-ir cell population appeared closeto the periventricular region, forming a band in the regionof the magnocellular preoptic nucleus (posterior parvocel-lular preoptic nucleus; Fig. 7H,I). Slightly more caudally,a conspicuous TH-ir cell population of rather large neu-rons with thick dendrites appeared just over the crossingof the optic tracts and extended to the postchiasmaticregion (Fig. 7I). This population corresponds to the supra-chiasmatic nucleus of Rink and Wullimann (2001).

DiencephalonIn the diencephalon and pretectum, various well-

dened nuclei and more scattered cell populations showedCR-ir perikarya. In addition, some conspicuous CR-irtracts and richly innervated areas of neuropil were ob-served. Some observations on the distribution of ancillarymarkers in relation to CR are also included.

Preglomerular nuclei and mammillary body. In thediencephalon, the largest accumulation of CR-ir perikaryawas found in the preglomerular nuclei-mammillary body,which extended from the ventrolateral postchiasmatic re-gion to the mammillary body bulging mediodorsally fromthe inferior hypothalamic lobes. The zebrash preglo-merular complex consists of several subdivisions, the mostrostral nuclei being the lateral (PGl), anterior (PGa), andmedial (PGm) preglomerular nuclei (Wullimann et al.,1996). CR immunohistochemistry clearly distinguishedbetween these nuclei: whereas most parts of the PGl werecomposed of cells without CR immunoreactivity, in thePGa a conspicuous cell band extending dorsomediallyshowed intensely CR-ir small cells (Figs. 3I,J, 8A). Thisband met the PGm, which had numerous cells with mod-erate to high CR immunoreactivity (Figs. 3K,L, 8B,C).

More caudally, the small-celled PGm was close to aventral nucleus of larger and more intensely CR stainedcells associated with more abundant neuropil (Fig. 8C),which appears to correspond to the tertiary gustatorynucleus (TGN) plus the subglomerular nucleus of Wulli-mann et al. (1996). Ventrocaudally, CR-ir cells extendedbetween the PGm and the mammillary body, which con-sisted of a large number of closely grouped, intenselyCR-ir small round neurons (Figs. 3N,O, 8D). NumerousCR-ir bers apparently originating from this complexcoursed rostrally in the lateral telencephalic bundle,which in the telencephalon split into a number of smallerCR-ir fascicles ascending to the Dm and neighboring pal-lial regions.

The preglomerular complex, including the tertiary gus-tatory nucleus of Wullimann et al. (1996), appeared toreceive very few or no ChAT-ir bers, in marked contrastto the inferior hypothalamic lobe (IHL) and torus lateralis(TLA; see below). Only the medial preglomerular nucleusappeared to be surrounded by ChAT-ir bers coursing tothe IHL and TLA; occasional ChAT-ir bers crossed thisnucleus. GAD immunocytochemistry revealed large differ-ences in innervation of the preglomerular-mammillarycomplex. The anterior and lateral preglomerular nucleidiffered in regard to the density and staining intensity ofGAD-ir terminals: GAD-ir boutons were very scarce in theCR-ir anterior preglomerular nucleus, in contrast to theadjacent lateral preglomerular nucleus (Fig. 8E). Themammillary body exhibited a characteristic GAD-ir neu-

ropil that extended rostrally to the medial preglomerularnucleus: specically, these nuclei contained a network ofneuropil cords with a number of GAD-ir boutons and -bers, among cords of densely packed GAD-negative cells(Fig. 8F). The tertiary gustatory nucleus plus the subglo-merular nucleus did not have this organization, but in-stead a rather homogeneous pale staining. The complexexhibited scant NPY-ir innervation and did not receiveGal-ir or TH-ir innervation.

Inferior hypothalamic lobe and torus lateralis. Inthe inferior hypothalamic lobe, two main regions could bedistinguished with CR immunocytochemistry, the diffusenucleus and the lateral recess nucleus (Fig. 9A). With CRimmunocytochemistry, the TLA appeared similar to thediffuse nucleus, and both nuclei contained a scatteredpopulation of intensely CR-ir small cells that were mostlylocated in supercial or intermediate regions (Figs. 3LN,9A,B). These regions had a large number of CR-ir beadedbers in most parts, with the neuropil of a similar denselystained appearance. In contrast, the dorsal part of theIHL, the central nucleus (located dorsocaudomedially tothe diffuse nucleus), contained scarce CR-ir bers (Figs.3M,N, 9A). The diffuse nucleus and TLA received numer-ous bundles of CR-ir bers: one of the tracts originatedfrom the strongly CR-ir secondary gustatory nucleus andapproached from caudal to rostral at the level of the trans-verse commissure, as observed in both transverse andsagittal thick sections (Fig. 3M). Another conspicuousbundle with CR-ir bers was the pretectomammillarytract of Wullimann et al. (1996), which approached theIHL rostrally.

The lateral recess nucleus (dorsal hypothalamus ofWullimann et al., 1996) was innervated by faintly stainedCR-ir bers, with the exception of its dorsomedial wall: inthis region, there was a rounded area that was very richlyinnervated by collateral bers from a small but intenselystained CR-ir bundle (Fig. 9A,D). This bundle apparentlyoriginated from a small raphe nucleus of the ventral mid-line of the trigeminal region containing densely stainedglobular cells (see the accompanying manuscript). It gaverise to a conspicuous thin bundle that coursed rostrally inthe brainstem, entering the IHL where it sent collateralsto the above-mentioned synaptic eld and going forwardthrough the ventrolateral region of the diencephalon todecussate close to the meninges, caudoventrally to thehorizontal commissure (Fig. 3IO). Here, this CR-ir fasci-cle is referred to as the raphe tract.

ChAT immunocytochemistry revealed a rich ChAT-irinnervation of the lateral recess nucleus, mainly in itsdorsolateral parts (Fig. 9E), and sparser but still signi-cant innervation of other parts of the IHL and the TLA,except for the central nucleus of the IHL, which practi-cally lacked ChAT-ir bers. A few pale ChAT-ir cells werefound inside this latter nucleus. A rather abundantChAT-ir innervation was observed in the lateral recessnucleus. Most of the IHL/TLA ChAT-ir innervation ap-peared to come from the very conspicuous ChAT-ir second-ary gustatory/visceral nucleus through a thick tertiarygustatory tract. In contrast, the IHL contained scatteredbut numerous strongly GAD-ir polygonal neurons, as wellas a mesh of GAD-ir bers and boutons, which was lessdense toward the lateral recess nucleus (Fig. 9F). Theregion of the lateral recess nucleus richly innervated bythe CR-ir raphe tract, however, exhibited scarce ChAT-irand GAD-ir innervation (Fig. 9E,F). NPY-ir innervation of


the IHL/TLA was sparse to moderate, and neither TH-irnor Gal-ir bers were present.

Medial hypothalamus and posterior tubercle. Themedial regions of the hypothalamus (the anterior tuberalnucleus, lateral hypothalamus, ventral hypothalamus,

lateral tuberal nucleus, and posterior hypothalamic lobe)showed scarce CR-ir structures: a few pale CR-ir smallcells were found in the ventral hypothalamus (anteriortuberal nucleus) and a few pale bers in the anteriortuberal nucleus (Figs. 3K, 10A). In the rostral part of the

Fig. 7. Photomicrographs of selected thin sections showing detailsof distribution of NPY (AC) and TH (DI) in the telencephalon andpreoptic area. A: Section of the precommissural telencephalon show-ing wide differences in NPY-ir innervation among the Dl, Dc, and Dp.Note also differences in innervation between dorsal and ventral partsof the Dl. Black arrows point to a row of negative cells at the limit ofDlDp; Open arrows indicate the approximate limit between the Dldand Dlv. B: Section showing differences in NPY-ir innervation be-tween the Dm and Dd. The arrows point to a row of negative cellsseparating these areas (the sulcus ypsiloniformis). C: Detail of thenumerous NPY-ir perikarya located in the Vl. D: Detail of the TH-ircell population of the central nucleus of the ventral area. E: Sectionthrough the postcommissural telencephalon showing the rich TH-ir

innervation of the region lateral to the Vp. F: Photomicrograph show-ing the fairly abundant TH-ir innervation of the Dp. G: Photomicro-graph of the bottom of the preoptic recess showing TH-ir neurons inthe vascular organ of the terminal lamina (solid arrow) and in theanterior parvocellular preoptic nucleus lateral to it (outlined arrow).Asterisk, blood vessel associated with the terminal lamina. Inset:Detail of TH-ir cells in the vascular organ. The arrowheads point toblood capillaries. H: Section of the posterior parvocellular preopticnucleus showing abundant TH-ir cells. I: Section through the supra-chiasmatic nucleus and caudal Ppp. Inset: Detail of a suprachias-matic TH-ir cell. In AF the midline is at the left. For abbreviations,see list. Scale bars 50 m in AG,I; 25 m in H, inset in G; 12.5 min inset in I.


Fig. 8. Photomicrographs of transverse sections through the pre-glomerular nuclei and mammillary body showing immunoreactivity tocalretinin (CR; AD) and GAD (E,F). A: Section showing CR-ir neu-rons in a part of the anterior preglomerular nucleus. B: NumerousCR-ir neurons are observed in the medial preglomerular nucleus, butCR-ir perikarya are limited to a few areas of the lateral preglomerularnucleus. C: This section through the medial preglomerular and thetertiary gustatory plus subglomerular nuclei shows obvious differ-

ences in staining intensity of CR-ir perikarya. D: Section of themammillary body showing that most perikarya are CR-ir. E: Differ-ences in GAD-ir innervation are observed between the anterior andlateral preglomerular nuclei. F: Section of the mammillary bodyshowing the characteristic pattern of GAD-ir innervation in irregularneuropil cords. Compare it with the innervation of the diffuse nucleus.In all gures, medial is to the left. For abbreviations, see list. Scalebars 50 m in AF.


Fig. 9. Photomicrographs of sections of the hypothalamic inferiorlobe and torus lateralis showing immunoreactivity to calretinin (CR;A,B,D), GAD (C,F), and ChAT (E). A: Section showing marked CRimmunoreactivity in the diffuse nucleus and torus lateralis. Note thatCR is practically absent from the posterior lobe and the lateral recessnucleus, except in a small portion of the latter. B: Detail of the toruslateralis showing CR-ir neurons (arrows). C: Section of the diffusenucleus showing GAD-ir neurons (arrows) and the abundance ofGAD-ir boutons except in the central nucleus. The large cells in theupper border are pigment cells. D: Detail of the section depicted in Ashowing the dorsomedial area of the lateral recess nucleus (outlinedarrow) receiving dense CR-ir innervation from the CR-ir raphe nu-

cleus tract (double arrow). Asterisk, caudal part of the medial preglo-merular nucleus; star, lateral recess. E: Section of the lateral recessnucleus showing dense ChAT-ir innervation in a region lateral to therichly CR-ir innervated area (outlined arrow). The broad arrow pointsto the bers of the tertiary gustatory tract. Asterisk, preglomerularnucleus; star, lateral recess. F: Detail of a similar section immuno-stained for GAD. Note that the CR-ir receiving region (outlined arrow)has very few GAD-ir boutons, which instead are numerous in otherparts of the lateral recess nucleus. Small arrow, GAD-ir neurons ofthe diffuse nucleus. Other symbols as in D and E. In all gures,medial is to the left. For abbreviations, see list. Scale bars 100 min A; 50 m in BF.

Fig. 10. Sections through the posterior tubercle (AF), epithala-mus (G,H), and thalamus (I) showing immunoreactivity to CR (A,G, I),TH (B,D,E), GAD (C), and ChAT (F,H). A: Section showing scarceCR-ir cells (arrowheads) in the posterior tuberal nucleus and theanterior tuberal nucleus (arrows). B: Large TH-ir neurons (arrow-heads) in the posterior recess nucleus. To facilitate comparison, theasterisk indicates the same region in A and B. C: Photomicrographshowing the very dense GAD-ir innervation of the posterior tuberalnucleus. D: Section just rostral to the lateral recess showing largenon-CSF-contacting TH-ir cells of the posterior tubercle (arrowheads)and numerous small CSF-c TH-ir cells (arrows) of the paraventricularorgan. E: Section of the transition between the posterior tubercle andposterior lobe showing numerous TH-ir cells of intermediate size

(arrow). F: Section through the posterior tuberal nucleus showing aChAT-ir cell (arrow) among small immunonegative cells. G: Sectionthrough the habenula showing a group of CR-ir ganglion cells (arrow)representing the parapineal organ. Note also a small band of CR-irbers (arrowhead) in the habenula. The midline is to the right. H: Sec-tion of the habenula showing the parcellation demonstrated by acompact band of small ChAT-ir cells (outlined arrows). I: Thick sec-tion passing through the periventricular pretectum and thalamusshowing the thalamic bands of CR-ir perikarya. The broad arrowpoints to the CR-ir pretectomammillary tract and nucleus. Note thickCR-ir bers in the posterior commissure. Star, third ventricle. Themidline is to the left. For abbreviations, see list. Scale bars 50 min AE,G; 12.5 m in F; 100 m in I.


posterior hypothalamic lobe a few CR-ir cells could beobserved near the posterior recess (Fig. 10A). The poste-rior lobe contained a few beaded CR-ir bers.

The posterior tubercle contained some CR-ir cells in theventrorostral region (the posterior tuberal nucleus) andextending laterally from the periventricular posterior tu-bercle ventral to the dorsal thalamic region. In the dorsalpart of the periventricular nucleus of the posterior tuber-cle of Wullimann et al. (1996) there were CR-ir cells withlong lateroventral dendrites (Figs. 3K,L, 10A). These cellsformed an oblique band extending laterally from periven-tricular regions. Other faintly CR-ir cells were alsopresent ventral and lateral to the band. In the posteriortuberal nucleus, faintly CR-ir cells with long dendritesdirected laterodorsally were also observed. Lateral to theperiventricular nucleus of the posterior tubercle and pos-terior tuberal nucleus, intermediate between these nucleiand the medial preglomerular nucleus, there were a smallnumber of medium-sized CR-ir neurons that formed acrown-like nucleus.

In the region of the posterior tuberal nucleus containingCR-ir cells, several large to very large TH-ir neurons couldbe observed (Fig. 10B; the periventricular nucleus of theposterior tubercle, Rink and Wullimann, 2001; group A11of Kaslin and Panula, 2001). The posterior tuberal nucleus(Fig. 10C) and posterior hypothalamic lobe were richlyinnervated by GAD-ir boutons, whereas the anterior tu-beral nucleus exhibited scarcer GAD-ir innervation. Fromthe posterior tuberal nucleus, a population of smallerTH-ir cells extended into the dorsal part of the posteriorhypothalamic lobe (group A9A10 of Kaslin and Panula,2001; diencephalic group 6 of Rink and Wullimann, 2002).Small cerebrospinal uid-contacting (CSF-c) TH-ir cellswere also present in the paraventricular organ just at thelevel of the lateral recess entrance (Fig. 10D) and associ-ated with the recesses of the posterior hypothalamic lobe,as noted previously (Kaslin and Panula, 2001; Rink andWullimann, 2002). Numerous small TH-ir cells were alsofound in the nucleus of the posterior hypothalamic recess(Fig. 10E). In the posterior tuberal nucleus, a few paleChAT-ir cells were also present (Fig. 10F). The posteriorlobe was richly innervated by Gal-ir bers and also con-tained a number of NPY-ir bers.

Thalamus. The medial region of the zebrash dien-cephalon contained CR-ir cell populations (Fig. 3HJ) dis-tributed in several of the thalamic nuclei of Wullimann etal. (1996), although sometimes their limits did not appearto match closely the limits of these authors nuclei. CR-ircells were found in both periventricular and more lateralregions, some of the latter cells forming apparentlyoblique lines extending from the periventricular regions(Fig. 10I). One of these lines of CR-ir cells could be ob-served in the anterior thalamic nucleus (in a posthabenu-lar region only); the CR-ir cells of the anterior thalamicnucleus formed a short band, dorsal to the ventral thala-mus, which appeared to lack CR-ir cell populations (Fig.3H). More caudally, both the central posterior and dorsalposterior thalamic nuclei contained many small faintlyCR-ir cells that appeared to extend lateroventrallythroughout a wide region just ventral to the fasciculusretroexus (Figs. 3I,J, 10H). In this region, CR-ir cellswere more abundant in the medial and dorsal borders ofthe dorsal posterior nucleus, and from its dorsal partCR-ir cells extended in the direction of the tractuspretecto-mammillaris of Wullimann et al. (1996). Pear-

shaped CR-ir cells were observed in central and ventralregions of the dorsal posterior thalamic nucleus, and thecaudal part of the dorsal thalamus also showed a band ofintensely CR-ir pear-shaped cells extending long den-drites laterally. The CR-ir cells of this caudal band weresomewhat larger and more intensely stained than those ofother thalamic regions.

In the dorsal thalamus, below the posterior commissure,there was a small population of small ChAT-ir cells (dorsalposterior nucleus). In addition, in the central posteriorthalamic nucleus there was a rather abundant populationof small NPY-ir cells. From this periventricular region,the NPY-ir population extended ventrolaterally towardthe region of the pretecto-mammillary tract, where ratherlarge NPY-ir cells were observed. The dorsal thalamuswas rather richly innervated by NPY-ir bers, mainly inits medial region. Ventral to the habenula and dorsal tothe preoptic nucleus, a ventral thalamic TH-ir cell popu-lation appeared; this population extended in the ventralthalamus caudally toward the posterior tubercle. A num-ber of GAD-ir boutons were also observed in the thalamus,mainly in periventricular and rostral parts. No Gal-irbers were observed, and only a few TH-ir bers coursedin the thalamus.

Pineal complex and habenula. In the meninges as-sociated with the posterior commissure there was a smallgroup of intensely CR-ir neurons that appeared to corre-spond to the parapineal organ (Figs. 3G, 10G), which islocated to the left of the pineal stalk (Gamse et al., 2003).In the pineal stalk, there were a few (three to ve) thickCR-ir bers that ran along most of its length. These bersentered the posterior commissure, but we were unable todetermine whether they originated in the brain or in thepineal organ.

Only one or two very pale CR-ir cells were observed inthe habenula, which contained scarce CR-ir bers, mainlyforming a loose band between the dorsal and ventral ha-benular regions (Fig. 3G, 10G). The habenula had a largeand compact population of small pale ChAT-ir neuronsthat formed a wide horizontal band rostrally and caudallywere restricted to the medioventral region (Fig. 10H), aswell as a small group of NPY-ir cells in the ventrolateralleft habenula. It also received some beaded NPY-ir bersand scarce TH-ir bers. Wide regions of the habenula,except for the region near the habenular commissure,lacked GAD-ir innervation.

Supercial pretectum. The zebrash supercial pre-tectum consists of several nuclei that have been describedby Wullimann and Meyer (1990) and Wullimann et al.(1996). The distribution of CR immunoreactivity in thesenuclei was characteristic (Figs. 3G,H, 11AC,FI). Nu-merous small strongly CR-ir neurons were present in theparvocellular supercial pretectal nucleus (PSP), mainlygrouped around a central neuropil that also showed somesmall CR-ir neurons and very numerous CR-ir processes(Figs. 3G, 11A,C,F). In transverse sections this is an ovalnucleus that makes a lateral prominence in the mostrostral region of the diencephalon, just below the optictectum. In sagittal sections, it appears as a cap closelyadjoining the optic tract rostrally. This nucleus gave riseto a conspicuous CR-ir commissural tract decussating inthe postoptic region (Figs. 3G,H, 11A,B; see below). GADand ChAT immunocytochemistry revealed additionalcharacteristics of the PSP. It contained a large populationof strongly GAD-ir cells, larger than the CR-ir cells, which


Fig. 11. Photomicrographs of sections passing through the super-cial pretectum showing immunoreactivity to CR (AC,FI), GAD(D,J), and ChAT (E,K). A,B: Thick sections showing a CR-ir commis-sural tract (black arrows) connecting the parvocellular supercialpretectal nucleus (PSP) and the neuropil of the dorsal accessory opticnucleus (white arrows). Note also the CR immunoreactivity of theoptic tract (outlined arrows). C: Detail of a thin sagittal sectionshowing numerous CR-ir cells and bers in the PSP. The nucleus isattached to the CR-ir optic tract. D: Transverse section showingGAD-ir neurons and bers in the PSP. Note that the optic tract isGAD-negative. E: Transverse section showing numerous ChAT-ir -bers in the PSP neuropil. FI: Series of transverse sections from

rostral to caudal showing the topographical relationships among thePSP, magnocellular supercial/posterior pretectal nucleus (PSM), ac-cessory pretectal nucleus (APC), and dorsal accessory neuropil (DAO).Note that the rim of CR-ir cells of the PSM (arrowheads) extendscaudally accompanying the pretectomammillary tract. J: GAD immu-noreactivity in the supercial pretectum. Note the similar appearanceof the GAD-ir distribution in the DAO and PSM, which is unlike thatobserved in both the APC and PSP. K: Section showing the ChAT-irinnervation of the APC, whereas ChAT-ir bers are practically lack-ing in the PSM and DAO. For other abbreviations, see list. Scalebars 250 m in A,B; 50 m in CK.

were mostly located peripherally, together with a richGAD-ir neuropil of rather uniform appearance (Fig. 11D).Moreover, this nucleus showed rich ChAT-ir innervationfrom bers coursing from the isthmus (Fig. 11E).

The pretectal supercial magnocellular nucleus, as seenin sagittal and transverse sections, consisted of a denserim of small CR-ir cells surrounding a central core mostlyfree of CR immunoreactivity (Figs. 3G,H, 11FH). Thecore and the rim were continuous caudally with the pre-tectomammillary tract of Wullimann et al. (1996) and itsaccompanying rim of CR-ir cells, respectively (Figs. 3I,J,11I). Rostral and caudal CR-ir cell populations of the PSMwere labeled by Wullimann et al. (1996) as PSM andposterior pretectal nucleus, respectively, but our resultssuggest that they form a single population. The carp PSMconsists of a cell rim and a glomerular neuropil core (Yo-shimoto and Ito, 1993) such as that observed here in thezebrash. As indicated above, the PSP gave rise to aconspicuous CR-ir ventral commissural tract coursing to astrongly CR-ir neuropil that was located ventrolaterally tothe PSM (Figs. 3H, 11A,B,F). This very rich CR-ir neuropilcorresponds to the dorsal accessory optic nucleus (DAO) ofWullimann and Meyer (1990). In addition to the verydense CR-ir neuropil, some CR-ir neurons could be ob-served in supercial parts. The PSM and DAO appeared tolack GAD-ir cells, but they were densely innervated byrather coarse GAD-ir boutons and received almost noChAT-ir bers (Fig. 11J,K). A number of GAD-ir cells wereseen in the rostral optic tectum in supercial layers, aswell as a number of coarse GAD-ir boutons located be-tween the fascicles of optic bers (themselves GAD nega-tive), suggesting that GAD-ir bers of the PSM/DAO couldoriginate from the neighboring optic tectum.

A further pretectal nucleus could be recognized medialand caudal to the PSP, consisting of rather large moder-ately CR-ir neurons and forming an oblique band corre-sponding to the accessory pretectal nucleus (APC) of Wul-limann and Meyer (1990). This nucleus is located betweenthe optic tectum and the PSM/posterior pretectal nucleus,from which it can be distinguished on the basis of thedifferences in CR immunoreactivity and neuron size (Figs.3H, 11H,I). The accessory pretectal nucleus received mod-erate ChAT-ir innervation and showed thinner andsparser GAD-ir boutons than those of the PSM/DAO (Fig.11J,K). These pretectal nuclei received scarce NPY-ir -bers and did not show Gal-ir or TH-ir bers.

Medial pretectum. In more central regions of the pre-tectum, CR immunohistochemistry revealed a scatteredgroup of medium-sized, pear-shaped CR-ir neurons thatextended lateral to the posterior commissure, just rostralto the torus semicircularis (Fig. 3I). This nucleus probablycorresponds to the medial pretoral nucleus reported inother teleosts. The posterior commissure itself containedsome thick CR-ir axons (Fig. 10I). In this region there wasa conspicuous TH-ir periventricular pretectal nucleuswith densely grouped small TH-ir neurons that projectedlateral processes, as reported previously (Rink and Wulli-mann, 2001; Kaslin and Panula, 2001). In the caudal partof the commissure, NPY-ir neurons were also observed.The regions containing the CR-ir and TH-ir cells had arather dense GAD-ir innervation.

Nucleus of the medial longitudinal fascicle. CR im-munocytochemistry revealed large pear-shaped CR-irneurons in the nucleus of the medial longitudinal fascicle(Nmlf), with about a dozen of these cells on each side (Figs.

3K,L, 12A,B). These cells were located in a shallow mid-line depression of the tectal ventricle, just rostral to theoculomotor nucleus. Both the cells and their dendriteswere included in a neuropil area rich in CR-ir dendritesand bers, and the perikarya were contacted by numeroussmall CR-ir boutons. Just below the nucleus, there was aconspicuous commissure of thick CR-ir bers (Fig. 12B).

DISCUSSIONIn the present study, the distribution of CR in the fore-

brain and the olfactory system of the adult zebrash wasinvestigated for the rst time, by using immunocytochem-ical techniques. Our results reveal that in this species CRis a specic marker of olfactory receptors and is widelyrepresented in various neuronal populations distributedthroughout the forebrain. In addition, CR immunocyto-chemistry reveals a characteristic pattern of bers andneuropil. Various centers in the telencephalon, dienceph-alon, posterior tubercle, and pretectal region contain oneor several CR-ir neuron types. Moreover, comparison ofthe distribution of CR observed in the forebrain of ze-brash (present results) with that reported in an ad-vanced teleost (Daz-Regueira and Anadon, 2000) revealsa number of similarities but also some interesting differ-

Fig. 12. Photomicrographs of thick transverse sections of the nu-cleus of the medial longitudinal fascicle (A,B) showing large CR-irneurons (arrowed). In the inset, the thick CR-ir commissural bers(arrowheads in B) are in focus. The open arrows in B and the insetindicate the same cell. Asterisks, medial longitudinal fascicle; stars,midbrain ventricle. Scale bars 50 m in A,B.


ences. This suggests that some aspects of the CR organi-zation have been maintained in different teleost evolution-ary lines, which raises the possibility that CR studies maybe useful for comparative analyses.

Olfactory systemOur results show that CR is expressed in olfactory re-

ceptor neurons (ORNs) of the zebrash, as well as in theolfactory nerves and olfactory glomeruli. The CR-ir ORNsare located throughout the olfactory epithelium, and theapical dendrites of these cells appear similar to those ofthe ciliary ORN type reported in zebrash (Byrd andBrunjes, 1995; Hansen and Zeiske, 1998). Interestingly,the microvillar ORNs and the recently described cryptolfactory neurons (Hansen and Zeiske, 1998) appear to beCR negative (present results). Crypt olfactory neuronsexpress another calcium-binding protein (S100; Germanaet al., 2004). These results suggest that expression of CRand other EF-hand calcium-binding proteins may be use-ful for characterizing major ORN subtypes in zebrash.Different odorants appear to elicit responses from differ-ent ORNs. For instance, microvillar ORN labeling appearsto be stimulated by amino acid odorants, whereas otherodorants fail to label these cells with the ion channelpermeant probe agmatine (Lipschitz and Michel, 2002).The use of immunocytochemical markers for different re-ceptor types, such as CR and other calcium-binding pro-teins, might help in deciphering the differential projec-tions of the major ORN types onto the olfactory bulbs.CR-immunoreactive ORNs have also been reported inother teleosts (Porteros et al., 1997; Daz-Regueira andAnadon, 2000), although the receptor type(s) has not beencharacterized.

Our results in zebrash suggest that the presence ofstrong CR immunoreactivity in glomeruli of the olfactorybulbs would indicate those receiving projections fromCR-ir ciliary ORNs. Our results reveal differences in sizeand staining intensity between CR-ir glomeruli of thedifferent glomerular elds, the largest glomeruli beingthose of the rostral and dorsolateral elds. Comparison ofthe present results with those of a DiI labeling study ofolfactory projections of zebrash (Baier and Korsching,1994) indicates that at least part of the dorsal glomeruli ofthese authors are either not or faintly CR immunoreac-tive, suggesting that they are innervated by other types ofORNs. Marked differences in CR immunoreactivityamong glomeruli have also been reported in vertebrates asdifferent as lampreys (Pombal et al., 2002), trout (Porteroset al., 1997), and rat (Bastianelli and Pochet, 1994), al-though the signicance of this fact is not known. Becausezebrash ORNs use calcium signals during activation(Friedrich and Korsching, 1997; Tabor et al., 2004), thepresence of calcium-binding proteins might be related tothe temporal characteristics of responses. Unlike in trout(Porteros et al., 1997), lamprey (Pombal et al., 2002), andmammals (Jacobowitz and Winsky, 1991; Malz et al.,2000; Alonso et al., 2001; Brinon et al., 2001; Kakuta et al.,2001), no CR-ir cells were detected in the zebrash olfac-tory bulbs.

Telencephalic lobesPrevious studies of the adult zebrash have used cyto-

architectonic criteria for delimiting telencephalic regions(Wullimann et al., 1996). CR immunocytochemistry re-vealed marked differences in immunoreactivity within

some cytoarchitectonic subdivisions of the telencephaliclobes of adult zebrash. In the dorsal area, outstandingCR expression was observed in bers innervating the pre-and supracommissural medial zone (Dm), but not the ros-tral Dm. The zebrash Dm is also heterogeneous dorso-ventrally, as shown by combinations of different markers,with clearly distinguishable dorsal and ventral parts (la-beled as 1 and 2, respectively, in Figure 4). Comparison ofthese areas with Figure 3C in Clemente et al. (2004)indicates that these parts are also distinguishable by ace-tylcholinesterase histochemistry and that the two are pal-lial.

Such immunohistochemical differences in the medialpart of the dorsal area are probably associated with nota-ble differences in their afferents and efferents, as observedin other teleosts with tract-tracing methods (Striedter,1991; Folgueira et al., 2004). Moreover, a population ofCR-ir neurons was also found in the dorsal Dm and Ddassociated with the sulcus ypsiloniformis at the level ofthe CR-ir ber-rich Dm region. Further pallial CR-ir cellpopulations were observed in the Dp and in the antero-ventral region of the dorsal area. These results indicatethat cytoarchitectonic subdivisions of the zebrash dorsalarea are not homogeneous as regards expression of CRand other immunocytochemical markers, as already re-ported for the trout dorsal area (Castro et al., 2003). More-over, the pattern of CR expression observed in the dorsalarea differs markedly among teleosts: notably, the Dm-DdCR-ir cell population of zebrash has not been observed introut or gray mullet, whereas the anterior Dd (Dd-a) CR-ircell population of trout does not appear to be present inzebrash or gray mullet (Daz-Regueira and Anadon,2000; Castro et al., 2003; present results).

The wide distribution of CR-ir cells observed in thezebrash subpallium recalls that observed in trout, al-though there are marked differences in the rostrocaudalextension of some populations: in trout, only the rostralVd contains CR-ir neurons (Castro et al., 2003), whereasin zebrash CR-ir neurons are found along most of therostrocaudal extension of the Vd (present results). A con-spicuous subpallial CR-ir cell population has also beenreported in the gray mullet (Daz-Regueira and Anadon,2000). The distribution of CR-ir cells in the zebrash pre-optic region and vascular organ of the terminal lamina isalso reminiscent of that observed in trout (Castro et al.,2003).

We also found marked regional differences in the pallialdistribution of the ancillary markers NPY, GAD, TH, andTRH, which may be useful for mapping the adult zebrashdorsal area. NPY-ir innervation was very high in thedorsal part of precommissural Dl, in the rostral Dm, andalso in the Dld at precommissural levels. In contrast, itwas very low in the Dp and moderate to low in the Dlv androstral Dd. This distribution in adult zebrash differssignicantly from that reported in another cyprinid spe-cies, the carp (Marchetti et al., 2000), in which the entiredorsal part of the dorsal area showed a large number ofNPY-ir terminals. In zebrash, there is also a very con-spicuous NPY-ir neuronal population in the lateral sub-pallium (Vl), as well as a smaller population in the Vd atprecommissural levels, which largely coincides with thatdescribed in the carp (Marchetti et al., 2000). In trout, thedorsal area was richly innervated by NPY-ir bers, but noclear zonation was observed, and NPY-ir cell populationswere observed in the Vl, Vd, and Vs (Castro et al., 1999).


Some regions of the dorsal area, including the posteriornucleus and, with lower intensity, the ventral region ofthe lateral part of the dorsal area (Dlv), exhibited moder-ate to abundant GAD-ir innervation. In the zebrash dor-sal area, TH immunoreactivity is found in some neuronsand bers of the caudal region, in an area in part corre-sponding to the posterior zone (Dp) of Wullimann et al.(1996). This TH-ir population is continuous with TH-irneuronal populations extending through the Vv, Vd, andVs, as previously reported by Rink and Wullimann (2001)and Kaslin and Panula (2001). In addition, some TRH-irbers project to a medial subzone of the Dm, as alreadyreported (Daz et al., 2002).

Finally, some regions appear to contain very low levelsof most of the markers used here: the central part of thedorsal area (Dc) can be dened as a zone that does notshow signicant CR, NPY, or TH immunoreactivity andonly exhibits scarce GAD-ir innervation. Acetylcholinest-erase histochemistry reveals large positive cells in thezebrash Dc (Clemente et al., 2004) and might be used asa positive marker of this region.

The subpallium is the zebrash telencephalic regionmost richly innervated by GAD-ir bers and is practicallythe only region to receive galanin-ir innervation, as in thetrout (Rodrguez et al., 2003). TH-ir structures observed inthe telencephalon of the zebrash are rather similar tothose reported by Rink and Wullimann (2001) and Kaslinand Panula (2001). The most important difference is thezebrash vascular organ of the terminal lamina, as re-vealed here for the rst time by both TH and CR immu-noreactivity. A very similar OVLT has been recognizedpreviously in trout and other teleosts (Gomez-Segade etal., 1991; Daz-Regueira and Anadon, 2000; Daz et al.,2001; Anadon et al., 2002; Castro et al., 2003). The OVLTappears to give rise to longitudinal tracts coursing cau-dally, as reported for the salmonid OVLT. Other TH-irgroups observed in the preoptic region are coincident withthose reported previously (Kaslin and Panula, 2001).

Two entopeduncular nuclei have been reported in theadult zebrash, the dorsal and ventral entopeduncularnuclei (Wullimann et al., 1996). Most of the so-called dor-sal entopeduncular nucleus may simply represent a cau-dal extension of the NPY-ir Vl nucleus, as indicated by thecontinuum of NPY-ir neurons with the same appearanceand organization found at precommissural, commissural(Vl), and postcommissural (dEnt) levels. This extended Vlnucleus comprising the dEnt region quite probablyprojects mostly to wide regions of the dorsal area, ratherthan to the habenula, which exhibits poor NPY-ir inner-vation. In the trout, the NPY-ir Vl population does notreach postcommissural levels (Castro et al., 1999), so inthis species there is no possibility of confusion with theentopeduncular nucleus, which projects massively to thehabenula (Yanez and Anadon, 1996). Observations withgeneral stains indicate that the ventral entopeduncularnucleus (vEnt) of the zebrash consists of a population ofdensely clustered small neurons that ascend clearly in thedirection toward the habenula, forming a prominent fas-cicle (unpublished observations), just like the entopedun-cular nucleus of the trout (Yanez and Anadon, 1996).Similar entopedunculo-habenular projections have beenreported in a cyprinid, the goldsh (Villani et al., 1996).

DiencephalonPreglomerular complex. A prominent CR-ir forma-

tion in zebrash is that formed by the preglomerular nu-clei and mammillary body. The PGm and the mammillarybody of this complex also share a similar pattern of GAD-irinnervation by coarse boutons among dense cords of smallneurons. In an advanced teleost, the gray mullet, thiscomplex extends to the midline in the postoptic region,roughly forming a circle. In the posterior tubercular re-gion, however, the strongly CR-ir caudal preglomerularcomplex can be clearly distinguished from the CR-negative mammillary body (Daz-Regueira and Anadon,2000), which is very different from that observed in ze-brash, in that the cells form a core around a wide centralneuropil, as in other percomorph species (Sawai et al.,2000).

Despite their homonomy, our CR results suggest thatthe zebrash mammillary body is not homologous to thatof percomorphs but rather to the caudomedial part of thepercomorph preglomerular nucleus. In this connection,the medial preglomerular nucleus of the zebrash is con-tinuous with the mammillary body, as also seen in trout(Folgueira et al., 2005), but no such continuity is seen inother cyprinids (goldsh, carp; see Braford and Northcutt,1983). Differences among cyprinid species might be due toeither organizational or nomenclatural differences. In thecarp, the medial preglomerular nucleus joins in the mid-line in the so-called commissural preglomerular nucleus(Braford and Northcutt, 1983), which suggests that cyto-architectural differences between zebrash and this spe-cies are genuine. The preglomerular nuclei/mammillarybody complex of zebrash appears to give rise to the con-spicuous CR-ir tract coursing in the lateral forebrain bun-dle and projecting to medial pallial regions (present re-sults), which would be consistent with both the heavyCR-ir projection to the Dm and the connections betweenthis complex and Dm traced experimentally in the trout(Castro et al., 2003; Folgueira et al., 2004, 2005). Thepreglomerular complex of carp appears to be part of sen-sory pathways afferent to the telencephalon from octavol-ateral centers (Murakami et al., 1986), whereas in troutthe complex receives telencephalic (Dm, Dc, Vv), tectaland toral bers and projects mainly to the telencephalon(Folgueira et al., 2004, 2005). The presence of CR immu-noreactivity in several nuclei of this complex in zebrashsuggests involvement of CR in specic sensory pathwaysprojecting to the ventral and dorsomedial telencephalon.

Inferior hypothalamic lobes. In the inferior hypo-thalamic lobes of zebrash, CR, ChAT, and GAD immu-nocytochemistry revealed several regions. First, the infe-rior lobes received rich CR-ir and ChAT-ir innervationfrom the secondary gustatory nucleus (SGN), via promi-nent fascicles. Because the lobes contain CR-ir neuronsand are richly innervated by other CR-ir ber systems,this SGN projection was only well dened with ChATimmunocytochemistry. Our results indicate that this tractheavily innervates the periventricular region of the lateralrecess at the level of and caudal to the entrance of thetract in the hypothalamus and more sparsely innervatesregions of the diffuse nucleus near these areas, in agree-ment with recent results in zebrash from other researchgroups (Clemente et al., 2004; Mueller et al., 2004). Ex-perimental tracing studies have demonstrated SGN pro-jections to the inferior lobe in goldsh and trout (Rink and


Wullimann, 1998; Folgueira et al., 2002, 2003). In theinferior hypothalamic lobes, CR clearly distinguishes thediffuse nucleus from the central nucleus, in regard to bothcell types and innervation.

A notable result is the highly specic innervation of aspecic dorsomedial portion of the lateral recess nucleusby a compact CR-ir tract that originates in neurons of theventral midline of the trigeminal region and coursesthrough the inferior lobes with a sigmoid pathway, de-cussating in the postoptic regions. The target region ofthis tract can also be differentiated from other parts of thelateral recess nucleus on the basis of its scarce innervationby both ChAT-ir and GAD-ir bers. As far as we areaware, no similar projection has been reported in anyother teleost. In the rainbow trout, the inferior hypotha-lamic lobes appear rather late in development as latero-caudal outgrowths originating from a single primitive hy-pothalamic lobe (Corujo and Anadon, 1990). Theapparently complex trajectory of this singular CR-ir tractof the zebrash may be the result of its early development,before the growth of the inferior lobes, in a similar way tothat seen in trout: deformations caused by differentialgrowth can be expected to affect the trajectory of thealready established tracts.

Thalamic nuclei. The zebrash dorsal thalamusshows some CR-ir cell populations, as reported in the graymullet (Daz-Regueira and Anadon, 2000). In the graymullet, these thalamic populations give rise to a conspic-uous CR-ir medial forebrain bundle that decussates in theregion of the anterior commissure (Daz-Regueira andAnadon, 2000); such a bundle has not been observed in thezebrash. Differences between these teleosts are also ob-served in the habenula, which in the gray mullet has aspecic CR-ir cell population that is not observed in ze-brash (Daz-Regueira and Anadon, 2000; present re-sults).

Supercial pretectum. The supercial pretectum ofteleosts is a complex region, with marked variations innuclear distribution among different groups of teleosts(Butler et al., 1991) and a lack of precise and comparableneurochemical and connectional studies in most of thespecies analyzed. Several pretectal nuclei have been de-scribed in the zebrash on the basis of cytoarchitectureanalyses, namely, the magnocellular supercial pretectal(PSM), parvocellular supercial, accessory, central, poste-rior, and DAO pretectal nuclei (Rupp et al., 1996; Wulli-mann et al., 1996); however, the identications of andlimits between these structures appear rather imprecise.CR immunocytochemistry seems to be a useful tool formore objective descriptions of nuclear structures in thesupercial pretectum of the zebrash, and reveals someother notable features. Two ancillary markers (GAD andChAT) have also helped in this characterization.

The PSP contains numerous small CR-ir neurons andoccupies a rostral position with respect to the optic tract.Its position corresponds to that of the nucleus of Wulli-mann et al. (1996). In addition, it contains rather abun-dant GAD-ir cells that have a somewhat different locationfrom the CR-ir neurons and receives a rich ChAT-ir inner-vation, as has been shown recently (Mueller et al., 2004).ChAT-ir bers probably originate from the nucleus isthmi,as reported in the trout (Perez et al., 2000). Projectionsfrom the nucleus isthmi to the PSP have also been dem-onstrated in the carp (Xue et al., 2001). Several of thesefeatures have been observed in the PSP of mullet (loca-

tion, CR-ir small neurons and neuropil) and trout (loca-tion, ChAT-ir neuropil; Perez et al., 2000), indicating con-served characteristics.

Our results indicate that the zebrash has a large ovoidnucleus, the magnocellular supercial/posterior pretectalnucleus (PSM), which appears to consist of a cellular rimsurrounding a wide central region of neuropil and bers.Different parts of this nucleus have been called the mag-nocellular supercial, central, and posterior pretectal nu-clei in the zebrash (Rupp et al., 1996; Wullimann et al.,1996). Under CR immunocytochemistry, the nucleus ap-pears as a single structure, and the cell rim shows ratherhomogeneous features with small to medium-sized CR-irneurons, fainter than those of the PSP. This organizationis similar to that reported in carp (Yoshimoto and Ito,1993). With electron microscopy, these authors describeda complex glomerular neuropil with two major types ofterminals, those with spherical synaptic vesicles andthose with attened vesicles. It appears probable that thenumerous thin GAD-ir boutons observed in zebrash cor-respond in part to those with attened vesicles, as re-ported in other vertebrate brain regions (Shepherd, 1998).There are also some GAD-ir small neurons, mainly in itsdorsomedial apex (the central pretectal nucleus).

In our view, the existing nomenclature in zebrash isconfusing, because several names are applied to the ros-tral, dorsal, and caudal levels of a single nucleus. On theother hand, the term central pretectal nucleus should bereserved for the cerebellopetal neuronal population dem-onstrated experimentally in several teleosts, including thecarp and the goldsh (Wullimann and Northcutt, 1988).Whether the zebrash central pretectal nucleus, whichconsists of -aminobutyric acid (GABA)ergic neurons(present results), projects to the cerebellum has not beeninvestigated.

Our results indicate that the dorsal accessory optic nu-cleus (DAO) of zebrash (Rupp et al., 1996; Wullimann etal. 1996) receives a major crossed projection from a con-spicuous CR-ir tract originating from the supercial pre-tectum, probably from the PSP, which gives rise to a verydense CR-ir neuropil. CR results in trout show thiscrossed pretecto-pretectal CR-ir tract and DAO neuropilmore clearly and reveal its origin from the PSP (unpub-lished observations); this crossed connection has also beenobserved in trout with DiI tract-tracing methods(Folgueira et al., unpublished observations). Our observa-tions suggest that in both the zebrash and trout the DAOis a specialized neuropil of the PSM/posterior pretectalnucleus that receives a crossed PSP CR-ir projection.

The present results show the same distribution ofGAD-ir bers in the PSM and DAO, without any discon-tinuity between them, which provides support for thishypothesis. Experimental studies in Lepomis has alsofound evidence of a bilateral projection from the PSP to anucleus similar to DAO termed the supercial pretectum,pars intermedia (Striedter and Northcutt, 1989). In gold-sh a prominent nucleus also termed the dorsal accessoryoptic nucleus (NAOD) has been shown to project to thecerebellum (Wullimann and Northcutt, 1988), but its cau-dal location in the diencephalon appears rather differentfrom the zebrash DAO. To add uncertainty, at present itis unknown whether the zebrash DAO projects to thecerebellum, or whether the goldsh NAOD receives acrossed projection from the PSP/PSM. A better knowledgeof the hodology of trout and zebrash DAO and of goldsh


NAOD appears to be necessary before any conclusionabout the homology of these nuclei can be reached.

CR immunocytochemistry in zebrash showed that theCR-ir cell rim of the PSM/posterior pretectal nucleus iscaudally continuous with a CR-ir cell population that em-braces the main nucleus output, the tractus pretectomam-millaris (TPM) of Wullimann et al. (1996), which can bereferred to as the nucleus of the TPM. This nucleusaccompanies the tract over a considerable distance. Insome cyprinids, the PSM/posterior pretectal nucleus re-ceives tectal afferents and projects to the mammillarybody and the nucleus lateralis valvulae (Northcutt andBraford, 1984; Yoshimoto and Ito, 1993), as it probablydoes in zebrash. The continuum of CR-ir cells observed inthe PSM/posterior pretectal nucleus and the nucleus ofthe TPM suggests a close functional relationship.

The zebrash accessory pretectal nucleus (APN) of Wul-limann et al. (1996) is a rather well-delimited nucleusconsisting of well-stained large CR-ir neurons and extend-ing dorsomedially between the PSM/posterior pretectalnucleus and the optic tectum. GAD immunocytochemistryalso shows homogeneous features of the neuropil through-out the nucleus. In view of its position, the group of largeCR-ir cells referred to as the PSM in mullet (Daz-Regueira and Anadon, 2000) probably corresponds to thezebrash APN. All these results suggest that CR-ir fea-tures of the pretectum may be well conserved in highlydivergent teleost lines.

CONCLUSIONSAnalysis of CR distribution in the zebrash forebrain

reveals 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 zebrash shows a largenumber of features shared with those observed in an ad-vanced teleost, the gray mullet, which indicates that theinvolvement of CR in functional circuits of the teleostbrain is probably rather conservative. Together, our re-sults indicate that CR is a marker of discrete cell popula-tions that appear to be well conserved throughout teleostsand that may be of value as a cell signature for decipher-ing complex brain morphogenetic processes in the teleostgroup.

LITERATURE CITEDAlonso JR, Brinon JG, Crespo C, Bravo IG, Arevalo R, Aijon J. 2001.

Chemical organization of the macaque monkey olfactory bulb: II. Cal-retinin, calbindin D-28k, parvalbumin, and neurocalcin immunoreac-tivity. J Comp Neurol 432:389407.

Anadon R, Rodrguez-Moldes I, Gonzalez A. 2002. Tyrosine hydroxylaseimmunoreactive neurons in the forebrain of the trout: organization,cellular features and innervation. Brain Res Bull 57:389392.

Andressen C, Blumcke I, Celio MR. 1993. Calcium-binding proteins: selec-tive markers of nerve cells. Cell Tissue Res 271:181208.

Arai R, Winsky L, Arai M, Jacobowitz DM. 1991. Immunohistochemicallocalization of calretinin in the rat hindbrain. J Comp Neurol 310:2144.

Baier H, Korsching S. 1994. Olfactory glomeruli in the zebrash form aninvariant pattern and are identiable across animals. J Neurosci 14:219230.

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