Distribution and functional organization of glomeruli in the olfactory bulbs of zebrafish (Danio...

Vol. 520 | No. 11August 1, 2012

The Journal ofComparative Neurology

Research in Systems Neuroscience

ISSN 0021-9967

Distribution and Functional Organization of Glomeruliin the Olfactory Bulbs of Zebrafish (Danio rerio)Oliver R. Braubach,1,2,3 Alan Fine,1,2* and Roger P. Croll1,2*1Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada2Neuroscience Institute, Dalhousie University, Halifax, Nova Scotia, Canada3Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea

ABSTRACTOdor molecules are transduced by thousands of olfac-

tory sensory neurons (OSNs) located in the nasal cav-

ity. Each OSN expresses a single functional odorant

receptor protein and projects an axon from the sensory

epithelia to an olfactory bulb glomerulus, which is

selectively innervated by only one or a few OSN types.

We used whole-mount immunocytochemistry to study

the neurochemistry and anatomical organization of glo-

meruli in the zebrafish olfactory system. By employing

combinations of antibodies against G-protein a subu-

nits, calcium-binding proteins, and general neuronal

markers, we selectively labeled various OSN types, their

axonal projections to glomeruli, and the detailed ana-

tomical distributions of individual glomeruli in different

regions of the olfactory bulb. In this way we identified

!140 glomeruli in each olfactory bulb of mature zebra-

fish. A small subset (27) of these glomeruli was unam-

biguously identifiable in nearly all animals examined.

These units were large and, located mainly in the

medial olfactory bulbs. Most glomeruli, however, were

comparatively small, anatomically indistinguishable, and

located in coarsely circumscribed regions; almost all of

these latter glomeruli were innervated by OSNs that

were labeled with anti-Ga s/olf and/or anti-calretinin

antibodies. Collectively, our results provide a uniquely

detailed description of a vertebrate olfactory system

and highlight anatomically distinct parallel neural path-

ways that mediate early aspects of olfactory processing

in the zebrafish. J. Comp. Neurol. 520:2317–2339,

2012.

VC 2012 Wiley Periodicals, Inc.

INDEXING TERMS: teleost; olfaction; immunocytochemistry; G-protein; calcium-binding protein; olfactory bulb

The olfactory system processes vital sensory informa-

tion related to feeding, habitat, conspecific status, and

predation. Odors are transduced into neural signals by

thousands (Buck and Axel, 1991) of different kinds of ol-

factory sensory neurons (OSNs), each of which expresses

only one functional odorant receptor (OR) type (Mom-

baerts, 2004; Serizawa et al., 2004). OSNs that express

the same OR type are scattered widely in the sensory epi-

thelia, but their axons project with great precision to syn-

aptic targets in the olfactory bulbs, the glomeruli. Each

glomerulus is innervated by one or at most a few OSN

types (Ressler et al., 1993; Vassar et al., 1994) and

responds to one or more potential odorants (Fuss and

Korsching, 2001; Mori et al., 2006). Glomerular activity

thus encodes odors in a spatial map of activation on the

olfactory bulb surface (Sharp et al., 1975; Moulton, 1976;

Stewart et al., 1979; Friedrich and Korsching, 1998; Vos-

shall et al., 2000; Wachowiak and Cohen, 2001) and the

output from this map is integrated in upstream brain

regions.

The olfactory systems of many insects are small and

contain relatively few glomeruli (!100), many of which

have already been catalogued in standardized anatomical

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: Natural Sciences and Engineering Research Council ofCanada Discovery Grants; Grant number: RGPIN170421 (to A.F.) and38863 (to R.P.C.); Grant sponsor: Canadian Institutes of Health ResearchOperating Grant; Grant number: MOP49489 (to A.F.); Grant sponsor: NovaScotia Health Research Foundation; Grant number: Predoctoral FellowshipMED-SRA-2006-5242 (to O.R.B.); Grant sponsor: World Class Institute(WCI)/National Research Foundation of Korea (KRF); Grant number: WCI2009-003; Grant sponsor: National Institutes of Health (NIH); Grantnumber: DC005259-39.

*CORRESPONDENCE TO: Alan Fine or Roger P. Croll, Department ofPhysiology & Biophysics, Dalhousie University, Halifax, Nova Scotia,Canada B3H 4R2. E-mail: [email protected] or [email protected]

These authors contributed equally to the study.

VC 2012 Wiley Periodicals, Inc.

Received September 20, 2011; Revised January 13, 2012; AcceptedFebruary 12, 2012

DOI 10.1002/cne.23075Published online February 17, 2012 in Wiley Online Library(wileyonlinelibrary.com)

The Journal of Comparative Neurology | Research in Systems Neuroscience 520:2317–2339 (2012) 2317

RESEARCH ARTICLE

atlases (Laissue et al., 1999; Galizia et al., 1999; Huetter-

oth and Schachtner, 2005; Masante-Roca et al., 2005;

Rybak et al., 2010). The availability of such atlases has

facilitated rapid progress in the physiological and molecu-

lar analysis of olfactory information processing in insects,

and certain types of behavior can now be directly linked

to the function of individually identifiable glomeruli (Suh

et al., 2004, 2007; Ibba et al., 2010). In contrast, map-

ping vertebrate glomeruli has proven far more challeng-

ing. Mice, for example, have large olfactory bulbs that

contain between 1,800 and 2,000 glomeruli (Mombaerts,

2006) and only a fraction of these are individually identifi-

able, and thus amenable to repeated study (Schaefer

et al., 2001; Potter et al., 2001; Jones et al., 2008; Oliva

et al., 2008).

An increasingly attractive alternative for studying the

neurobiology of vertebrate olfaction is the zebrafish,

Danio rerio. This species is advantageous for this purpose

because it is economical, small, genetically manipulable,

and suitable for in vivo optophysiological experiments.

Furthermore, each zebrafish olfactory bulb reportedly

contains only 80 glomeruli (Baier and Korsching, 1994),

and these are segregated into distinct functional sub-

groups known to process different types of odors. For

example, amino acids (food odors) are processed by a

distinct group of lateral glomeruli (Friedrich and Korsch-

ing, 1997), while bile acids and pheromones are proc-

essed by glomeruli in the medial olfactory bulbs (Friedrich

and Korsching, 1998). Despite the apparent simplicity of

the zebrafish olfactory system, a detailed and reproduci-

ble anatomical map of individual glomeruli has not yet

been established (but see Baier and Korsching, 1994).

Therefore, the purpose of the present study was to map

zebrafish glomeruli in greater detail than had been previ-

ously attempted. In order to achieve this goal we estab-

lished a state of the art whole-mount immunohistochem-

istry method and employed a series of general and

specific immunocytochemical markers for OSN axons to

describe and catalog glomeruli in all regions of the olfac-

tory bulbs.

MATERIALS AND METHODSAnimals

Wildtype zebrafish (AB strain: University of Oregon)

were maintained at the Aquatron facility at Dalhousie Uni-

versity according to standard guidelines (Nusslein-Vol-

hard and Dahm, 2002). The fish were kept at 27.5"C on a

12/12-hour light/dark schedule in 10-L holding tanks

(Aquatic Habitats, Apopka, FL) continuously supplied with

a drip of fresh dechlorinated water, which was passed

through a series of biofilters and a charcoal filter. The fish

were fed several times daily with staple fish food (Omega

Sea, Sitka, AK) and live nauplii of Artemia (Salt Creek,

Salt Lake City, UT). All experiments were conducted on

adult zebrafish that were 3 months of age or older and

between 2–3 cm in length. A total of 26 zebrafish were

used to create the images and numerical data shown.

Animals were handled according to guidelines estab-

lished by the Canadian Council for Animal Care.

Tissue preparationZebrafish were killed by immersion in cold water

(<4"C for 1 minute) and decapitated. The dorsal cranium

was removed and exposed brains were fixed by immer-

sion in fresh 2% paraformaldehyde (PFA; Electron Micros-

copy Sciences, Hartfield, PA) in phosphate-buffered sa-

line (PBS: 100 mM Na2HPO4, 140 mM NaCl, pH 7.4) for 6

hours at room temperature, or overnight at 4"C. The fish

were next washed five times over 2 hours in PBS before

dissecting the heads further to isolate the forebrain and

olfactory epithelia; these were then placed in a PBS-

based blocking solution containing 0.25% Triton X-100,

2% dimethyl sulfoxide, 1% bovine serum albumin, and 1%

normal goat serum (PBS-T; all from Sigma, St. Louis, MO)

for #12 hours at 4"C. Unless noted otherwise, PBS-T was

used for all subsequent wash steps, each of which com-

prised minimally five rinses over a period of !4 hours.

Antibody characterizationWe employed a combination of antibodies (Table 1) to

label OSN axons and their synaptic terminals, both of

which are abundant in vertebrate glomeruli (Kasowski

et al., 1999; Nezlin et al., 2003). To label glomeruli nonse-

lectively, we used an antibody against keyhole limpet he-

mocyanin (KLH), which was previously used in zebrafish

and trout as a marker for an unknown epitope in or on

OSN axons (Riddle and Oakley, 1992; Fuller et al., 2006;

Gayoso et al., 2011). We also counterstained some prep-

arations with anti-synaptic vesicle protein 2 (SV2), which

Abbreviations

dG Dorsal glomerulusdlG Dorsolateral glomerulusIR Immunoreactive / immunoreactivitylG Lateral glomeruluslP Lateral plexusmaG Medial anterior glomerulusmdG Mediodorsal glomerulusmpG Medial posterior glomerulusOB Olfactory bulbOE Olfactory epitheliumON Olfactory nerveOR Olfactory receptorOSN Olfactory sensory neuronvaG Ventroanterior glomerulusvmG Ventromedial glomerulusvpG Ventroposterior glomerulus

Braubach et al.

2318 The Journal of Comparative Neurology |Research in Systems Neuroscience

labels axon terminals in the core of glomeruli (Koide

et al., 2009).

To characterize the functional organization of glomer-

uli, we employed antibodies against G-protein a subunits

and calcium-binding proteins. These proteins are selec-

tively expressed in certain OSN types and thus permit dis-

crimination of their somata and axonal projections into

the olfactory bulbs (Hansen et al., 2003; Germana et al.,

2007; Gayoso et al., 2011). Anti-Ga s/olf (Table 1), which

recognizes a 42–45 kDa protein in the olfactory epithe-

lium of lampreys (Frontini et al., 2003) and catfish (Han-

sen et al., 2003), was used to label ciliated OSNs and

axons, as demonstrated previously in zebrafish (Koide

et al., 2009; Gayoso et al., 2011). We also tested anti-

Gaq/11 and anti-Gao (Table 1), which label microvillous

and crypt cells in catfish (Hansen et al., 2003) and anti-

Gai-3 as an additional marker for microvillous receptor

neurons (Hansen et al., 2005). To our knowledge, none of

these antibodies were used previously in zebrafish, and

we thus performed control experiments to confirm their

specificity (below).

We also used anti-calretinin (Table 1), which recognizes

a 28–29 kDa protein in zebrafish (Castro et al., 2006;

Germana et al., 2007) as another marker for ciliated cells

and their axonal projections (Castro et al., 2006; Gayoso

et al., 2011). Finally, we used an antibody against the

calcium-binding protein S100 (Table 1), which recognizes

a 10 kDa protein in zebrafish (Germana et al., 2007;

Gayoso et al., 2011) to label crypt and/or ciliated OSNs

and their axons.

The antibodies employed in this study were used previ-

ously, and under similar experimental conditions, in

zebrafish and related species (citations above). We there-

fore conducted preadsorption controls only for antibodies

for which we had no prior reference. These were rabbit

anti-Gao, anti-Gai-3, and anti-Gq/11 antibodies (Table 1). A

synthetic peptide was obtained for each antibody (Santa

Cruz Biotechnology, Santa Cruz, CA) and added at 200

lg/ml to 1:200 dilutions of primary antibody solution

(see below), preadsorbed for 24 hours at room tempera-

ture and then spun for 10 minutes at 1,500g. Several

fixed brains were incubated in the supernatant and proc-

essed as described below. None of these brains exhibited

immunolabeling. We also conducted negative controls

by processing brains without incubation in primary anti-

bodies; none of the specimens exhibited detectable

fluorescence.

Whole-mount immunocytochemistryMixtures of primary antibodies were diluted 1:50–

1:200 in PBS-T (Table 1) and brains were incubated in

these solutions for at least 7 days at 4"C with gentle agi-

tation. Longer incubation periods (3–4 weeks) improved

staining and were performed when possible. After incuba-

tion in primary antibody solutions, brains were washed

and then incubated in a mixture of secondary goat anti-

rabbit antibodies (for polyclonal antibodies) and goat anti-

mouse antibodies (for monoclonal antibodies) conjugated

to either Alexa Fluor 488 or Alexa Fluor 555 (both from

Invitrogen, Burlington, ON, Canada). All secondary anti-

bodies were used at a final dilution of 1:100 in PBS-T and

brains were incubated in these solutions for 5–7 days

at 4"C.

Before mounting, all brains were rinsed three times in

PBS-T and then an additional three times in PBS alone.

The brains were then immersed in a 3:1 solution of glyc-

erol to 0.1 M Tris buffer (pH 8.0) containing 2% n-propyl

gallate (all from Sigma) for a minimum of 24 hours for

clearing. Afterwards, the tissue was mounted in fresh

glycerol solution between coverslips separated with

stacks of coverslip fragments. Slides were then sealed

with nail polish and stored at 4"C prior to being viewed

with a confocal microscope (below).

TABLE 1.

List of Antibodies, Their Sources and Staining Protocols

Antibody Immunogen/Host Source

General labels (1:100 in PBS-T)Anti-keyhole-limpet-hemocyanin(KLH) (1:100 dilution)

keyhole limpet hemocyanin/rabbit Sigma (H0892)

Anti-synaptic vesicle protein 2(SV2) (1:100 dilution)

Diplobatis ommata synaptic vesicles/mouse Developmental Studies HybridomaBank (Iowa City, IA, USA)

OSN type specific Labels (1:50-200 in PBS-T)Anti-Ga s/olf (1:50 dilution) rat c-terminus (aa: 377-394)/rabbit Santa Cruz Biotech (Santa Cruz,

CA, USA) sc-383Anti-Ga o (1:50 dilution) rat divergent domain (aa: 105-124)/rabbit Santa Cruz sc-387Anti-Ga i-3 (1:50 dilution) rat c-terminus (aa: 345-354)/rabbit Santa Cruz sc-262Anti-Gaq/11 (1:50 dilution) mouse common domain (aa: 115-133)/rabbit Santa Cruz sc-392Anti-calretinin (1:200 dilution) recombinant human calretinin/mouse Swant (Bellinzona, Switzerland) 6B3Anti-S100 (1:100 dilution) purified cow S100 protein/rabbit Dako (Glostrup, Denmark) Z 0311

Olfactory Glomeruli in Zebrafish

The Journal of Comparative Neurology | Research in Systems Neuroscience 2319

Immunocytochemistry on cryosectionsOlfactory epithelia were removed from fixed heads (4%

PFA for 12 hours) and immersed in 20% sucrose in PBS

for 12 hours. Isolated epithelia were then placed in

embedding medium (Tissue Tek Optimal Cutting Temper-

ature Medium, Fisher Scientific, Ottawa, ON, Canada),

quickly frozen, and cryosectioned at a thickness of 8 lm.

The sections were collected on gelatin-coated slides and

air-dried for 30 minutes before being postfixed in 4% PFA

for another 30 minutes. After three washes in PBS con-

taining 0.3% Triton X-100 (0.3% PBS-T), sections were

blocked in 0.3% PBS-T containing 10% normal goat serum

for 30 minutes.

Mixtures of primary antibodies (Table 1) were diluted

1:500 in 0.3% PBS-T and placed directly onto each tissue

section; the slides were then transferred to a humid

chamber and incubated overnight at room temperature.

The next day, sections were rinsed three times with PBS-

T (20 minutes each) and solutions of appropriate second-

ary goat antibodies conjugated to Alexa Fluor dyes (1:250

dilution; Invitrogen) were placed on top of the sections

and left for 5 hours at room temperature. Sections were

then washed four times with 0.3% PBS-T before being

mounted in fresh glycerol mounting medium (see above)

and then coverslipped.

Axon tracingTo study the organization of OSN axon terminals in

the lateral plexus, we carried out anterograde tracing

with the lipophilic tracer 1,10-dioctadecyl-3,3,30,30-tetra-

methyl-indo-carbocyanine (DiI 5 mg/5 mL in dimethyl

formamide; Invitrogen). Fish were decapitated and fixed

in 4% PFA overnight at 4"C; after several rinses in PBS

heads were dissected to expose the olfactory epithelia

and olfactory nerves. We then dipped the tips of fine for-

ceps into a drop of tracer solution and gently squeezed

the olfactory nerves with the forceps in order to apply the

dye. Specimens were then transferred to 0.5% PFA (in

PBS) and stored in a dark chamber at 37"C. Under these

conditions, the tracer spread to the olfactory bulbs within

48 hours. At this time, we removed any nonneural tissue,

placed the brains in a drop of PBS, and coverslipped the

tissue.

Microscopy and image processingSpecimens were imaged with a Zeiss LSM 510 META

laser scanning confocal microscope (Carl Zeiss, Thorn-

wood, NY) and serial optical sections were obtained from

the olfactory bulbs at 1-lm intervals to a maximum depth

of 100 lm. To achieve optimal visualization of glomeruli,

we imaged each olfactory bulb individually using a 25$immersion objective (Zeiss LCI Plan-Neofluar); olfactory

bulbs were thus imaged separately in each specimen and

composite images of bilateral pairs were created after-

wards (see below). Olfactory bulbs were also imaged

from multiple sides (i.e., dorsal, lateral, ventral, and

medial) to obtain high-resolution image data of glomeruli

on each olfactory bulb surface.

Optical sections were viewed in ImageJ (http://rsb.in-

fo.nih.gov/ij) and photomicrographs were created by

superimposing stacks of optical sections to a depth indi-

cated in each figure panel. Separate color channels were

merged in ImageJ and RGB composite images were then

processed further in Photoshop (Adobe Systems, San

Jose, CA). Specifically, we adjusted intensity and contrast

levels to set the darkest pixel to full black and the bright-

est pixel to full white in each image. Occasionally, granu-

lar background staining in areas outside of the olfactory

bulbs was minimized via noise filtering (e.g., despeckled).

Images were then cropped, rotated, and assembled into

composite figures and each figure was exported to Adobe

InDesign and added to figure plates. All diagrams accom-

panying the figures were created with Adobe Illustrator.

Identification of glomeruliA structure was tentatively considered a glomerulus if

it was roughly spheroidal and encircled by OSN axons

that formed an anatomically distinct stalk (s) connected

to the olfactory nerve. In specimens that were counter-

stained with synaptic labels, these structures had to

colocalize with an aggregate of SV2-immunoreactive (IR)

puncta indicative of the synaptic neuropil that comprises

the glomerular core (Manzini et al., 2007). We conducted

a series of preliminary experiments to determine if differ-

ent labels consistently represented the same numbers

and distributions of glomeruli. In doing so, we identified

anti-KLH and anti-SV2 as the most reliable structural

markers for glomeruli in all areas of the olfactory bulb,

while anti-calretinin was useful for labeling glomeruli in

some, but not all, regions (see Results). Other antibodies

(Table 1) did not produce equally detailed labeling of glo-

meruli, but were nevertheless useful to determine the

neurochemistry of glomeruli.

Glomeruli were counted and measured by stepping

through optical sections and tracing outlines of their axo-

nal innervation with the freehand drawing tool in ImageJ.

All measures were obtained from single optical sections

in which the maximal circumferences of individual glo-

meruli were detected. Average counts and sizes of glo-

meruli were based on observations from at least four fish

of each sex (see Table 2 and below); these averages were

obtained separately for left and right olfactory bulbs but

did not differ in preliminary comparisons. The data cited

in this study are thus pooled measurements from left and

right olfactory bulbs.

Braubach et al.


TABLE

2.

SummaryofNumbers,

Sizes,an

dInnervationofGlomeruliin

theMature

Zebrafish

Olfactory

System

GroupGlomerulus

Glomeruliper

Bulb

Area(lm

2)

Innervation

PreviousDescriptionname

Dorsalglom

eruli

$(n

%7)

#(n

%4)

$#

Baier

andKorsching,1994;Gayosoet

al.,2011;Satoet

al.,2005

(zebrafish);Frontini

etal.,2003(lamprey)

dG1

11

7426

81

7136

70

calretinin

notidentified

dGx

14.6

63.5

16.2

62.7

3846

70

4886

100

Ga

s/olf/calretinin

anterior

plexus

(noglom

eruliidentified)

Dorsolateralglom

eruli

$(n

%8)

#(n

%6)

$#

Baier


al.,2011;Satoet

al.,2005(zebrafish)

dlG1

11

9326

394

10006

494

Gas/

olf

dorsal

clusterassociated

glom

eruli1-5

(zebrafish)

dlG2

0.7

60.5

0.7

60.5

8286

242

10526

329

Gas/

olf

dlG3

0.9

60.3

0.7

60.5

8386

396

11636

504

Gas/

olf

dlG4

0.7

60.5

0.8

60.3

8786

337

12636

510

Gas/

olf

dlG5

10.7

60.4

11396

572

13036

510

Gas/

olf

dlGx

49.6

64.5

44.3

64

2526

82

3606

88

Ga

s/olf/calretinin

glom

eruliof

thedorsal

cluster(zebrafish)

Ventrom

edialglom

eruli

$(n

%10)

#(n

%4)

$#

Baier


al.,2011;Satoet

al.,2005

(adu

ltzebrafish)

vmG1

0.8

60.4

19156

233

10656

231

Ga

s/olf/calretinin

Ventral

tripletglom

eruli1-3

and/

orventromedialglom

erulus

notidentified

vmG2

0.8

60.4

19626

246

9886

264

Ga

s/olf/calretinin

vmG3

10.8

60.4

13386

467

9636

441

Ga

s/olf/calretinin

vmG4

11

10706

263

12746

58

calretinin

vmG5

0.7

60.5

18846

287

7126

328

calretinin

notidentified

vmG6

0.8

60.4

112516

409

13426

439

Ga

s/olf/calretinin

notidentified

vmG7

0.8

60.4

0.8

60.4

17396

320

19326

325

Gas/

olf

notidentified

vmGx

76

2106

19816

315

8286

63

Gas/

olf

notidentified

vmGy

146

2146

23696

67

3046

38

Ga

s/olf/calretinin

notidentified

Ventroanteriorglom

eruli

$(n

%7)

#(n

%6)

$#

Baier


al.,2011(zebrafish);Frontini

etal.,

2003(lamprey)

vaGx

76

1.3

76

2.3

4806

140

4046

90

Ga

s/olf/calretinin

ventroanterior

glom

eruli/anterior

plexus

(zebrafish),ventralcluster(lamprey)

Ventrop

osterior

glom

eruli

$(n

%10)

#(n

%6)

$#

Baier


al.,2011;Satoet

al.,2005(zebrafish,

goldfish)

vpG1

0.9

60.3

123976

857

31166

927

KLH

notidentified

vpG2

11

22016

547

22966

878

KLH

vpG

Lateralglom

eruli

$(n

%7)

#(n

%4)

$#

Baier


al.,2011;Satoet

al.,2005(zebrafish)

lG1

0.1

60.3

0.5

60.6

1796

15096

154

calretinin

glom

erulus

1of

thelateralchain(lcG1)

lG2

11

29406

151

37836

557

Gas/

olf

lcG2

lG3

11

21486

517

25786

275

calretinin

lcG3

lG4

11

31906

512

40856

675

calretinin

lcG4

lG5

0.3

60.4

0.256

0.5

14806

783

2012

calretinin

lcG5

lG6

11

27976

429

26996

406

calretinin

lvpG

lGx

126

2.4

106

28786

224

7766

195

calretinin

notidentified

Mediodorsalglom

eruli

$(n

%8)

#(n

%6)

$#

Baier


al.,2011;Satoet

al.,2005(zebrafish)

mdG

11

112776

341

13966

653

KLH

mediodorsal

posteriorglom

erulus

1(m

dpG1)

mdG

21

122166

644

23506

730

S100

mdpG2

mdG

30.9

60.2

113566

346

18096

630

KLH

notidentified

mdG

41

115546

591

19016

846

KLH

notidentified

mdG

51

113806

243

15096

458

Gao

notidentified

mdG

60.9

60.2

0.7

60.5

18006

438

18446

313

KLH

notidentified

Medialanterior

glom

eruli

$(n

%7)

#(n

%4)

$#

Baier

andKorsching,1994;Satoet

al.,2005(zebrafish)

maG

11

113996

393

11546

399

Ga

s/olf/calretinin

maG

inadult

maG

x17.8

63.6

16.8

63.3

3436

110

3236

117

Ga

s/olf/calretinin

notidentified

Medialpo

steriorglom

erulus

$(n

%7)

#(n

%4)

$#

Baier


al.,2011(zebrafish)

mpG

11

15626

477

24576

364

KLH

medialelongatedglom

erulus



Glomerular nomenclature and classificationThe glomerular nomenclature used here is based on

previous schemes used in developing (Dynes and Ngai,

1998) and adult zebrafish (Baier and Korsching, 1994).

However, we modified the glomerular nomenclature in

several instances to provide consistency among these

previous reports and our results. Specifically, we

assigned glomeruli to certain groups based on their loca-

tion on one of the four olfactory bulb surfaces (i.e., dorsal,

ventral, lateral, medial groups). Glomeruli of each group

were then assigned names based on their anatomical

location with respect to other groups on each olfactory

bulb surface (e.g., ventroanterior and ventromedial glo-

meruli). Glomeruli that could be unambiguously identified

in at least 70% of specimens were given a specific num-

ber (e.g., ventromedial glomerulus vmG1); if a glomerulus

had been numbered previously, we tried to retain its origi-

nal numbering even if it was not identifiable in 70% of

specimens (e.g., lG5). The remaining glomeruli were

assigned to appropriate regions (e.g., ventromedial glo-

merulus vmGx) and discriminated from anatomically or

neurochemically dissimilar units where necessary (e.g.,

ventromedial glomerulus vmGy). We compare our nomen-

clature to previously used schemes in Table 2.

Statistical analysisNumbers and sizes of glomeruli were pooled according

to their identity (e.g., individually identifiable dG1) or the

region in which they were located (e.g., indistinguishable

dGx) across specimens (female vs. male). Data are pre-

sented as means and their standard deviations throughout

this report. To determine if differences in glomerular size

were statistically significant, we compared pooled anatomi-

cal data from individually identifiable or anatomically indis-

tinguishable glomeruli via a one-way analysis of variance

(ANOVA). Possible differences in anatomical variability

among numbers of different types of glomeruli were eval-

uated with Levene’s test for equality of variances; these

consisted of the pooled variances for all individually identi-

fiable glomeruli throughout the olfactory bulbs and the sep-

arate variances among anatomically indistinguishable glo-

meruli within certain identifiable groups (e.g., all dlGx). If

significance was observed in Levene’s test, we further eval-

uated variances among certain groups of glomeruli through

post-hoc Student’s t-tests. All statistics were analyzed with

SPSS software (Chicago, IL).

RESULTSDifferent OSN types and topography ofaxonal projections into the olfactory bulbs

The zebrafish olfactory system consists of bilaterally

symmetric olfactory epithelia (OE in Fig. 1A) that are

folded into rosette-shaped sensory organs; each OE is

connected via a short olfactory nerve (ON in Fig. 1A) to

the olfactory bulbs (OB in Fig. 1A). The olfactory epithelia

contain distinct OSN types, which were distinguishable

based on their locations in the sensory epithelium, their

morphologies, and their labeling with antibodies against

G-protein a subunits and calcium-binding proteins. Cili-

ated OSNs were abundant and easy to distinguish from

other cells (Fig. 1B); they displayed round somata located

deep in the epithelium and extended long, ciliated den-

drites to the epithelial surface (see also Castro et al.,

2006; Gayoso et al., 2011). Ciliated OSNs were labeled

reliably with the anti-calretinin antibody (Fig. 1D) and also

displayed anti-Ga s/olf IR; the latter was observed primar-

ily in the cell membrane and dendritic knobs on the epi-

thelial surface (Fig. 1C).

OSNs at intermediate depths in the olfactory epithe-

lium displayed various morphologies and differences in

antibody labeling. For example, OSNs labeled with the

anti-Gao antibody had elongated perikarya and each bore

a single short dendrite (Fig. 1F). Small apical processes

were often visible at the tips of these dendrites (arrow-

head in Fig. 1F). We observed similar OSNs in anti-calreti-

nin and anti-S100 labeled tissue (arrow in Fig. 1E); these

cells also displayed small processes at the tips of their

dendrites (arrowhead in Fig. 1G). However, at the imaged

resolution it was not possible to identify intermediate

cells as either microvillous or ciliated.

Crypt OSNs were identifiable based on their ovoid

shape, rounded apical pole, eccentric basal nucleus, and

location near the surface of the sensory epithelium. Cells

that displayed this morphotype were labeled by the anti-

S100 antibody (Fig. 1H) and were scattered across the ol-

factory epithelium. Occasionally we also observed pre-

sumptive crypt OSNs in tissue labeled with the anti-Gao

antibody (asterisk in Fig. 1F).

Finally, two other antibodies against G-protein a subu-

nits (anti-Gai-3 and anti-Gaq/11) did not produce unambig-

uous and/or reproducible labeling in the OE or olfactory

bulbs (not shown). Labeling with the anti-KLH antibody

also resulted in inconsistent labeling of OSN somata (not

shown), but reliably labeled OSN axons (below).

Neurochemistry and anatomicalorganization of glomeruli

OSN axons targeted 1406 8 and 1456 6 glomeruli in

the each olfactory bulb of adult female (n % 7) and male

zebrafish (n % 4), respectively. The glomeruli were organ-

ized in nine distinct regions reproducibly located on dor-

sal, ventral, lateral, and medial surfaces of the olfactory

bulbs. We summarize the organization of olfactory glo-

meruli in mature zebrafish in schematic maps in Figure 2

and discuss our findings in more detail below.

Braubach et al.


Figure 1. Organization of the zebrafish olfactory system and olfactory sensory neurons (OSN) types in the sensory epithelium. A: A maximum in-

tensity confocal projection of a whole-mounted zebrafish olfactory system stained with an anti-calretinin antibody and viewed from the ventral side.

The olfactory epithelia (OE) are connected to the olfactory bulbs (OB) via a short olfactory nerve (ON). B: The anti-calretinin antibody labels ciliated

OSNs, which have round somata (s) and slender dendrites (d) that terminate in bundles of cilia (c) on the epithelial surface (see also detailed view in

D). Ciliated OSNs also display anti-Ga s/olf IR in the plasma membrane and in ciliated knobs that cover the surface of the olfactory epithelium (C). F:

Anti-Gao labels cells located at intermediate depths in the sensory epithelium; such cells display short dendritic protrusions, on which small proc-

esses are occasionally visible (arrowhead in F). Similar cells were also labeled by anti-calretinin and anti-S100 antibodies (arrows in E, G). Anti-S100

also labeled ovoid crypt cells with accentric nuclei (n) located near the epithelial surface (H). Dashed lines in C-H indicate the basal lamina. Images

in A,B depict whole-mounted tissue, and images in C-H are from cryosectioned epithelia. Scale bar in H applies to C-F also.



Figure 2. Schematic maps summarizing the distribution of glomeruli. Glomeruli are arranged in nine groups on the dorsal (A), ventral (B),

lateral (C), and medial (D) olfactory bulb surfaces. Each panel depicts a single right olfactory bulb viewed from a different perspective. Glo-

meruli that could be identified and mapped as distinct individuals across all or most specimens are given specific names and numbers

(e.g., dG1 in A) while the remaining glomeruli, which could not be identified as individuals but instead could only be recognized as units

belonging to certain glomerular clusters, are denoted with an ‘‘x’’ or a ‘‘y’’ (e.g., dGx in A). The labeling of glomeruli with specific neuro-

chemical markers is indicated in color (see legend). Figure references to representative examples for different glomeruli are provided

where applicable.

Braubach et al.


Dorsal groupsDorsal glomeruli (dG)

We detected !15 dG in the anterodorsal olfactory bulb

(Fig. 2A), a region in which no glomeruli were previously

described (see Table 2). Within this region the dorsal glo-

merulus 1 (dG1) was individually identifiable and distin-

guishable from its surroundings because of its strong

staining with the anti-calretinin antibody (dG1 in Fig.

3A,C,D) and its stereotypic position near the posterior

edge of the dG cluster (compare dG1 in Fig. 3C,F). We

were able to identify this glomerulus in all specimens

examined (Table 2).

The remaining dorsal glomeruli (dGx) uniformly dis-

played KLH-like IR (Fig. 3B), calretinin IR (Fig. 3C), and Ga

s/olf IR (Fig. 3E) and were not individually identifiable.

Throughout the remainder of this article nonidentifiable

glomeruli are denoted by an ‘‘x or y’’ and/or traced with

dashed outlines (e.g., see dGx in Fig. 4C). In comparison

Figure 3. A,D: Maximum intensity confocal projections of dorsal olfactory bulbs labeled with different antibody combinations (as indi-

cated). The dorsal (dG), dorsolateral (dlG), and mediodorsal (mdG) glomerular groups are traced with dashed lines in A,D and individually

identifiable glomeruli are traced with solid lines and are numbered throughout (e.g., dlG1 in B). B: The anti-KLH antibody uniformly labels

the OSN axons that innervate the dorsal olfactory bulbs, permitting detection of many dorsal glomeruli. C,F: Anti-calretinin and (E)

anti-Gas/olf antibodies label the dG and dlG but not the mdG. Certain glomeruli displayed selective or uneven IR for one antibody but not

another (e.g., dlG1-5 in B,C). The depth of each confocal projection is indicated in C and F. A magenta/green variant of the image in A is

provided in Supporting Figure 1A. Scale bars in C,F apply to each column.



to the dG1 (above), the dGx showed considerable anatom-

ical variations within and between specimens. For exam-

ple, the numbers of detectable dGx ranged from 10–21

and 14–20 glomeruli in female (n % 7) and male (n % 5)

zebrafish, respectively. Furthermore, the dGx also varied

in sizes and shapes, having cross-sectional areas ranging

from 175 lm2 (arrowheads in Fig. 4C) to 512 lm2 (arrow

in Fig. 4B). On average, however, the dGx were smaller

than the individually identifiable dG1, which appeared to be

a common distinction between identifiable and nonidentifi-

able glomeruli in all areas of the olfactory bulbs (below).

Finally, the dG occasionally displayed IR with the anti-S100

antibody (e.g., Fig. 10A). However, this observation was

inconsistent and we could not replicate this finding in sev-

eral of our specimens; we therefore omitted inconsistent

anti-S100 labeling (e.g., dG) from our schematic glomerular

maps in Figure 2.

Dorsolateral glomeruli (dlG)The dlG (Fig. 2A) were tightly clustered on the dorsolat-

eral olfactory bulb surface of both female and male zebra-

fish (Figs. 3A, 4A). The dlG comprised approximately one-

third of the total glomerular population (Table 2); however,

only five dlG were individually identifiable based on their

unique neurochemistry, morphology, and position. Specifi-

cally, the dlG1-5 displayed KLH-like IR (Fig. 3B) and Ga s/olf

IR (Fig. 3E), but never calretinin IR (compare dlG1-5 in Fig.

3B,C). Furthermore, the dlG1–5 were significantly larger

than the dlGx (P < 0.001, ANOVA; Table 2) and reproduci-

bly arranged as a row of five units along the caudal edge of

the olfactory bulbs, !40–60 lm beneath the surface of

the olfactory bulb (see dlG1–5 in Fig. 4D).

The dlGx were comparatively small and uniformly dis-

played KLH-like IR (Fig. 3B), calretinin IR (Fig. 3C), and Ga

s/olf IR (Fig. 3E). The dlGx also displayed occasional labeling

with the anti-S100 antibody (e.g., Fig. 10A); however, this

was not consistent and thus not included in our maps in

Figure 2. Numerical variations were common among the

dlGx but were not as profound as elsewhere (e.g., dGx dis-

cussed above) and the dlGx had a largely homogeneous

appearance (dlGx in Fig. 4A,B). Occasionally, we found dlGx

that resembled one another in the left and right olfactory

bulbs of single animals (arrowheads in Fig. 3B), but such

similarities were not recognizable in other animals. Thus,

based on the staining used in this study only a fraction of

dlG could be repeatedly identified in the olfactory bulbs of

mature zebrafish.

Ventral groupsVentromedial glomeruli (vmG)

The vmG consisted of diverse glomeruli (Fig. 2B),

with average cross-sectional areas ranging from

Figure 4. Identifiable and nonidentifiable glomeruli in dorsal groups. A: Maximal intensity confocal projection of the dorsal olfactory bulbs

(50 lm depth). B–D: Partial projections of 10–20 1-lm-thick serial optical sections (depth indicated). The outlines of some glomeruli are

traced with solid and dashed lines to indicate individually identifiable (solid) and indistinguishable glomeruli (dashed). All dorsal glomeruli

label uniformly with the anti-KLH antibody. The dorsolateral glomeruli 1–5 are individually identifiable and are arranged in a row (see dlG1–

5 on the right side of D). Most dlG are anatomically indistinguishable from one another and cannot be identified as individual units (see

dlGx in A and traced dlGx in B,C). The dorsal glomeruli (dGx) are anatomically diverse and vary in shapes and sizes (compare arrow in B to

arrowheads in C); only a single unit, dG1 (C), can be repeatedly identified in the dorsal group. The mediodorsal glomeruli (mdG) are large

and each is individually identifiable. Scale bar in D applies to all panels.

Braubach et al.


304 6 38 lm2 (vmGy in Table 2) to 1,932 6 325

lm2 (vmG7 in Table 2). The innervation of all vmG dis-

played KLH IR (Fig. 5B) but additional labeling with

anti-calretinin (Fig. 5C) and anti-Ga s/olf antibodies

(Fig. 5E) revealed neurochemical differences among

these glomeruli.

Figure 5. A,D: Maximum confocal projections of ventral olfactory bulbs labeled with different antibody combinations (as indicated). The

tissue is the same as shown in Figure 3, but was rotated and imaged from a ventral perspective. B: The anti-KLH antibody labels the

innervation of ventral glomeruli, including the ventroanterior (vaG), ventromedial (vmG), and ventroposterior (vpG) groups. C,F: The anti-cal-

retinin antibody incompletely labeled the vmG (e.g., compare vmGx in B,C) but reliably labeled glomeruli in the lateral group (see lG in

C,F). In contrast, Ga s/olf IR axons targeted the vmG but not the lG (D,E). Glomerular clusters are traced with dashed lines in A,B, and indi-

vidually identifiable glomeruli are traced with solid lines. The depths of confocal projections shown in each column are indicated in C and

F. A magenta/green variant of the image in A is provided in Supporting Figure 1B. Scale bars in C,F apply to each column.



Figure 6

Braubach et al.


The most consistently identified vmG were vmG1–7,

which were comparatively large (Table 2) but variably

stained by the anti-calretinin and anti-Ga s/olf antibodies

(see vmG1–7 in Fig. 6A–C,E–G). The vmG1–3, for example,

were proximal to each other, strongly calretinin IR (see

vmG1–3 of different specimens in Figs. 5C,F, 6E), and of-

ten had an area devoid of staining at their center (e.g.,

vmG1,3 in Fig. 6A). Additional vmG were located posteri-

orly to vmG1–3 (see vmG4–6 in Fig. 6A,F). These units were

labeled selectively with either the anti-calretinin (see

vmG4–5 in Fig. 6A,B) or anti-Ga s/olf antibodies (see vmG6

in Fig. 6A,B). In contrast, the vmG7 was located anteriorly

to the above-mentioned units and always labeled selec-

tively with the anti-Ga s/olf antibody (see vmG7 in Fig.

6C,F,G). Overall, we identified the vmG1–7 in more than

70% of specimens (Table 2). We detected additional, ana-

tomically similar glomeruli in the proximity of vmG1–7

(arrowheads in Figs. 5C, 6A,B) but detection of these

units varied between specimens and they were not unam-

biguously identifiable.

Another distinct group of ventral glomeruli, the vmGx

(see Fig. 2B), was located !10–50 lm from the ventral

surface of the olfactory bulb. These glomeruli were

selectively stained with anti-KLH (see vmGx in Fig. 6B)

and anti-Ga s/olf antibodies (see vmGx in Fig. 6F) and

were located anywhere between the posteroventral and

anteroventral regions of the olfactory bulb. It was not

possible to unambiguously identify the vmGx because

they displayed varying distributions and numbers in the

olfactory bulbs of different animals.

Finally, the vmGy comprised a cluster of compara-

tively small glomeruli that were located near the mid-

line between the two olfactory bulbs (Figs. 2B, 5C).

These glomeruli were always labeled intensely with

anti-calretinin antibody (vmGy in Figs. 5C, 6E), and dis-

played reliable but weaker KLH IR (compare vmGy in

Fig. 5B,C) and Ga s/olf IR (compare vmGy in Fig. 5E,F).

The vmGy were among the smallest glomeruli in the

olfactory bulbs, and to our knowledge had not been

previously identified, even though they comprise a sig-

nificant number of glomeruli in both female and male

zebrafish (Table 2).

Ventroposterior glomeruli (vpG)We identified two large glomeruli in the ventroposterior

olfactory bulbs (see vpG1–2 in Figs. 2B, 5B). The vpG were

among the largest and most consistently identifiable glo-

meruli in both female and male zebrafish (Table 2); how-

ever, with the immunohistochemical labels employed

here it was not possible to resolve the neurochemistry of

the innervation of either vpG. Both units could therefore

only be visualized in specimens that were stained with

nonselective labels such as anti-KLH (Fig. 5B) and anti-

SV2 antibodies (not shown).

Ventroanterior glomeruli (vaGx)A small group of vaGx was located near the olfactory

nerve entry, in the ventroanterior olfactory bulbs (Fig.

2B). This region consists of many defasciculating axon

bundles that project to other areas, and previous descrip-

tions have suggested that this region is aglomerular.

However, we typically identified seven small glomeruli

(Table 2), located 11–20 lm beneath the superficial axon

bundles (Fig. 6D,H). The vaGx labeled consistently with

anti-KLH (vaGx in Fig. 6D) and anti-Ga s/olf (vaGx in Fig.

6H) antibodies but inconsistently with anti-calretinin anti-

body (see Fig. 6H). The vaGx differed conspicuously in

sizes, shapes, and distributions, and were not individually

identifiable.

Lateral groupLateral glomeruli (lG)

The lateral olfactory bulb surface (Fig. 2C) consisted of

morphologically diverse glomeruli and a diffuse aggregate

of OSN axons, the lateral plexus (lP). The lG and the lP

were labeled strongly with anti-KLH (Fig. 7B) and anti-cal-

retinin antibodies (Fig. 7C,F), sparsely with the anti-Ga s/

olf antibody (Fig. 7E), and occasionally with the anti-S100

antibody (not shown).

Five large lG (lG1–5) were located along the dorsal edge

of the lateral group (Table 2). The lG1 and lG5 were only

occasionally identifiable, as they were surrounded by a

diffuse aggregate of OSN axons: in two lateral olfactory

bulbs shown in Figure 8, for example, only the lG1 or the

lG5 was visible in either specimen (lG1 in Fig. 8E and lG5

Figure 6. Complexity in the distribution and neurochemistry of ventromedial glomeruli. A–D: Subprojections of optical sections through

the ventral glomerular layer; the specimen is the same as that shown in Figure 5A. E–H: Subprojections through the ventral glomerular

layer in another specimen labeled with anti-calretinin and anti-Ga s/olf antibodies. Ventromedial glomeruli consist of individually identifiable

ventromedial glomeruli 1–7 (see vmG1–7 in A,C), which label selectively with certain antibodies (e.g., see vmG6 in A). The vmGx are selec-

tively anti-KLH IR (B) and anti-Ga s/olf IR (E). A group of comparatively small vmGy is located near the midline separating the olfactory

bulbs (see vmGy in A,B). The ventroanterior glomeruli (vaGx) consist of small units that are located proximal to the olfactory nerve entry

(D,H). The largest ventral glomeruli are the ventroposterior glomeruli (see vpG in A). The depth of each subprojection is indicated. Ma-

genta/green variants of the images in A-D are provided in Supporting Figure 2. Scale bars in D,H apply to each column.



in Fig. 8B). The lG2, which was located on the caudal edge

of the lateral group, was identifiable in every specimen

(lG2 in Figs. 7A,D, 8A,D; Table 2). The lG2 was further dis-

tinguishable because it was the only lateral glomerulus

that was innervated by Ga s/olf IR but not calretinin IR

axons (compare lG2 in Fig. 7E,F). The lG3 (Fig. 8A,D) and

lG4 (Fig. 8C,F) were also identifiable in all animals exam-

ined (Table 2) but, unlike lG2, these glomeruli displayed

calretinin IR but not Ga s/olf IR (compare lG3 in Fig. 7E,F).

Because of their large size and stereotypic arrangement,

however, these glomeruli were easily identifiable (Table

2). Finally, lG6 was located on the ventroposterior edge of

Figure 7. (A,B) Maximum intensity confocal projections of lateral olfactory bulbs labeled with different antibody combinations. The speci-

mens are the same as shown in Figures 3 and 5. Most lateral glomeruli (e.g., lG5 in B and lG3 in C,F) are innervated by calretinin IR axons

and do not display Ga s/olf IR (E). The lG2, however, is innervated by Ga s/olf IR, but not by calretinin IR axons (indicated in F). The postero-

lateral olfactory bulb surface appears to be devoid of glomeruli and instead consists of a lateral plexus (lP). The lateral glomerular cluster

is traced with dashed lines in A,D and individually identifiable glomeruli are traced with solid lines throughout the figure. A magenta/green

variant of the image in A is provided in Supporting Figure 1C. Scale bars apply to all panels within each column, and the depth of the con-

focal projections is indicated at the bottom of C,F.

Braubach et al.


the lateral group and was best visualized from a ventral

perspective (Fig. 5C,F).

Additional lateral glomeruli (lGx) were smaller than the

lG1–6 (Table 2; P < 0.05, one-way ANOVA) and varied in

shapes, sizes, and locations between olfactory bulbs of

the same and different animals (compare lGx in Fig. 8A–

C and 8D–F). The lGx were located either along the ven-

tral edge of the lateral group (lGx in Fig. 8F and from a

Figure 8. Morphological diversity of lateral glomeruli (lG) in olfactory bulbs from two zebrafish. A,D: Maximum intensity confocal projec-

tions (depth indicated) of right lateral olfactory bulbs. B,C,E,F: Stepwise subprojections of 2–9 serial optical sections (1-lm intervals) pro-

duced from the same data. The lG1–5 are large units located along the dorsal edge of the lateral group. The lG1 (E) and lG5 (B) can only

occasionally be resolved, while other large glomeruli (lG2–4) are always visible in the positions shown throughout A–C and D–F. Variable-

sized lGx (dashed outlines) are present in every animal, but cannot be individually identified. The lateral plexus (LP) is a diffuse aggregate

of axon termini (arrows and arrowheads in G) that appears to be devoid of glomeruli proper. Note that OSN innervation shown in G is la-

beled with a lipophilic tracer DiI. Scale bars in C and F apply to each column unless indicated otherwise.



ventral perspective in Fig. 5C) or below the lateral olfac-

tory bulb surface (see Fig. 8B,C). The lGx were present in

every animal examined, but we could not discriminate

them from one another with the labels employed in this

study.

Finally, the posterolateral olfactory bulbs were occu-

pied by a fibrous lateral nerve plexus (lP in Fig. 8A,D),

which appeared to be devoid of glomeruli proper and

instead consisted of axon termini, which were indiscrim-

inately arranged (Fig. 8G). These axon termini occasion-

ally clustered and resembled small glomerular struc-

tures (arrowheads in Fig. 8G), but more often

resembled varicosities and/or swellings on the shafts

or endings of axons (arrows in Fig. 8G). Occasionally

we also observed weaker calretinin IR in the lP than in

areas containing the lG (e.g., lP in Figs. 7C, 7F).

Medial groupsMediodorsal glomeruli (mdG)

Six mdG were arranged in a roughly triangular, bilater-

ally symmetric cluster on the dorsomedial olfactory bulb

surface (Fig. 2A,D). The mdG were often stacked on top

of one another, which made it difficult to discern all six

units from a single view plane (e.g., dorsal view in Figs.

3B, 4C). However, if the bulbs were viewed from their

Figure 9. Maximum intensity confocal projections of the medial olfactory bulb surface labeled with different antibody combinations. The tis-

sue is the same as in Figures 3, 5, and 7 but was rotated and imaged from a medial perspective. The medial anterior glomerulus 1 (maG1 in

B,C,E,F) is identifiable in every animal. The remaining maGx are small and variably innervated by calretinin IR and Ga s/olf IR axons; only some

maGx are labeled by the anti-calretinin antibody while Ga s/olf IR is visible in all maGx (compare arrowheads in E,F). The medial posterior glomer-

ulus (mpG) was visible in nearly all specimens examined, but only if these were labeled with the anti-KLH antibody (see mpG in B and compare

to E,F). The mediodorsal glomeruli (mdG) are labeled with the anti KLH antibody (mdG in B) but display no calretinin IR or Ga s/olf IR. Visible glo-

merular clusters are traced with dashed lines in A,D and individually identifiable glomeruli are traced with solid lines and numbered. A ma-

genta/green variant of the image in A is provided in Supporting Figure 1D. Scale bars in each column apply to all panels.

Braubach et al.


medial surface, all mdG could generally be identified (Fig.

9B), and by imaging our specimens from multiple angles

we could identify six mdG in almost all animals (Table 2).

All mdG displayed uniform KLH IR (Figs. 3B, 9B) but only

some labeled additionally with other neurochemical

markers. Specifically, the mdG2 was innervated by S100

IR axons (Fig. 10A) in all specimens that we studied, and

the mdG5 displayed reliable labeling with the anti-Gao

antibody (Fig. 10B).

Medial anterior glomeruli (maG)The maG were located on the medial olfactory bulb sur-

face, near the olfactory nerve entry (Fig. 2D), and were

best visualized in bulbs that were lying on their medial

side (Fig. 9). Only the maG1 was reliably identifiable. Like

other individually identifiable units, the maG1 was compa-

ratively large (Table 2), but unlike most other individually

identifiable glomeruli, maG1 displayed both calretinin IR

(Fig. 9C,F) and Ga s/olf IR (Fig. 9E).

The remaining maGx were among the smallest glomer-

uli in the olfactory bulbs (Table 2) and were uniformly la-

beled by the anti-KLH antibody (maGx in Fig. 9B). Further-

more, all maGx uniformly displayed Ga s/olf IR (Fig. 9E) but

were only partially visible in specimens that were labeled

with an anti-calretinin antibody (compare arrowheads in

Fig. 9E,F). Similar to other small glomeruli, the arrange-

ments of maGx varied between animals, and we could

thus not identify them as individual units.

Medial posterior glomerulus (mpG)The mpG (Fig. 2D) was located between ventromedial

and medial olfactory bulb surfaces. This unit was only visi-

ble in specimens that were stained with the anti-KLH anti-

body (compare Fig. 9B with Figs. 9C,E,F). The mpG was

generally elongated and was innervated by a single thick

axon bundle; as with most other large glomeruli, the mpG

was identifiable in every specimen.

Stereotypy versus variability of identifiableand indistinguishable glomeruli

From the data presented above we conclude that 27

glomeruli can be identified in at least 70% of olfactory

bulbs from the same (i.e., left and right) and different

zebrafish (Table 2). Most of these glomeruli labeled exclu-

sively with an antibody against a G-protein a subunit or

calcium-binding protein (e.g., lG2 in Fig. 7D), but not with

both (with the exception of some vmG and maG1 as

shown in Fig. 2). Alternatively, some glomeruli were only

labeled with the anti-KLH antibody (gray glomeruli in Fig.

2). Furthermore, the 27 individually identifiable glomeruli

were comparatively large, and their average maximal

cross-sectional area of 1,859 6 698 lm2 was signifi-

cantly greater than that of remaining glomeruli (pooled

average 512 6 285 lm2; P < 0.001, ANOVA). Finally, all

individually identifiable glomeruli were stereotypically

positioned in certain areas of the olfactory bulbs, as

described above. In contrast, the remaining 82% of glo-

meruli could not be identified as individuals but were only

recognizable as units belonging to certain regions. These

glomeruli almost always labeled with antibodies raised

against Ga s/olf and calretinin and, as stated above, dis-

played significantly smaller cross-sectional areas than

individually identifiable units.

All glomeruli displayed some degree of variation in

prevalence, but data presented in Table 2 indicate that

Figure 10. Selective innervation of two mediodorsal glomeruli. Shown are dorsal views of two whole-mounted olfactory bulbs. The mdG2

is targeted by S100 IR axons (A), which occasionally also appear to innervate the remaining dorsal glomeruli; however, while S100 IR is

highly consistent for the innervation of mdG2 it cannot be reliably observed for other dorsal glomeruli. B: The mdG5 is the only glomerulus

that is innervated by Gao IR axons. The outline of other glomeruli can be seen based on anti-SV2 labeling (e.g., dlG in A). Dorsal glomeru-

lar clusters are traced with dashed lines but individual mdG tracings were omitted for clarity.



the numbers of individually identified glomeruli are less

variable than those of anatomically indistinguishable

units. To test the significance of this observation we com-

pared the pooled variance among the 27 individually iden-

tifiable glomeruli to that of the Ga s/olf IR and calretinin IR

glomeruli in the dG, vmG, maG, vaG, and lG groups. An

overall comparison between these variances was indeed

statistically significant (P < 0.05, Levene’s test), and fur-

ther comparisons indicated that the variability of the dGx,

vmGx, maGx, and lGx was significantly greater than that of

the 27 individually identifiable glomeruli (all P < 0.05,

Student’s t-test). Thus, glomeruli in the zebrafish olfactory

system differ in several specific anatomical features, and

by their degree of anatomical stereotypy and variability.

DISCUSSIONWe used zebrafish to create what is, to our knowledge,

one of the most detailed anatomical descriptions of the

glomerular organization in any vertebrate (see also Baier

and Korsching, 1994; Gaudin and Gascuel, 2005). We

estimate that there are over 140 glomeruli in each olfac-

tory bulb of adult zebrafish, regardless of sex. These glo-

meruli can be classified as either of two types. The first

type consists of 27 large glomeruli that are nearly invari-

ant in number and arrangement and thus individually

identifiable. These glomeruli are furthermore distinguish-

able by their selective labeling with antibodies raised

against calcium binding proteins or G-protein a subunits.

The other glomeruli (82% of total) are smaller on average

and are located in broadly circumscribed regions of the

olfactory bulbs; these units cannot be distinguished from

one another and were almost always labeled with both

anti-calretinin and anti-Ga s/olf antibodies.

Identification and detectionof new glomeruliIdentification strategy

The sensory input to a glomerulus consists of axons

and terminals from hundreds (Hildebrand and Shepherd,

1997) to thousands of OSNs (Mombaerts, 2006), and the

convergence of these axons onto discrete spheroidal loci

is a fundamental feature of olfactory system anatomy

(Chen and Shepherd, 2005). Labeling OSN axons and

their terminals with anatomical or functional probes is

therefore an established method to study the structure,

distribution, physiology, and development of olfactory glo-

meruli (Friedrich and Korsching, 1998; Bozza et al., 2004;

Feinstein et al., 2004; Li et al., 2005; Sato et al., 2005;

Wachowiak et al., 2009; Koide et al., 2009; Bellmann

et al., 2010). In the present study we defined a glomeru-

lus as a roughly spheroidal aggregate of axon shafts and

terminals. Furthermore, each glomerulus had to be sepa-

rately innervated by axon bundles connected to the olfac-

tory nerve and had to be separated from nearby units by

areas devoid of labeling.

Using a similar identification strategy, Baier and

Korsching (1994) previously identified 80 olfactory glo-

meruli in adult zebrafish. Among these were 22 stereo-

typically arranged glomeruli, most of which we could also

identify. For example, both Baier and Korsching (1994)

and we identified a row of five glomeruli in the dorsopos-

terior olfactory bulbs (dlG1–5 in Fig. 2A). Furthermore, our

descriptions of anatomically indistinguishable glomeruli

also correspond well, and we identified 44–50 dlGx in the

same region in which Baier and Korsching (1994) previ-

ously identified 49 glomeruli. Some of the glomeruli

described here and in Baier and Korsching (1994) have

also been detected with genetic OSN axon labels (Sato

et al., 2005) or with indicators of neural activity (Friedrich

and Korsching, 1998). Thus, identifying glomeruli based

on their OSN innervation produces valid and consistent

results.

Detection of new glomeruliWe are the first group to employ whole-mount immu-

nohistochemistry combined with exhaustive confocal

imaging of all olfactory bulb surfaces to study the distri-

bution of zebrafish glomeruli. Our approach produced

anatomical data with unprecedented anatomical detail

and enabled us to identify significantly more glomeruli

(!140) than were previously known (!80). For example,

we identified !15 glomeruli (dGx) in the anterodorsal ol-

factory bulb; this area was previously described as a fi-

brous, aglomerular nerve plexus in zebrafish (Baier and

Korsching, 1994; Gayoso et al., 2011) and related spe-

cies (Kosaka and Hama, 1982; Frontini et al., 2003). We

believe that our ability to detect these glomeruli was

due to both the improved labeling obtained with certain

antibodies and our whole-mount imaging approach.

Indeed, even the smallest glomeruli were clearly identifi-

able in tissue that was labeled with anti-KLH or anti-cal-

retinin (e.g., vmGy in Fig. 5B,C). However, the same glo-

meruli were difficult to visualize in tissue stained with

anti-Ga s/olf (vmGy in Fig. 5E) or lipophilic axon tracers

(pers. obs.); both of these markers had been used in

previous descriptions of the fish olfactory system (Baier

and Korsching, 1994; Frontini et al., 2003; Hansen

et al., 2003; Gayoso et al., 2011). Furthermore, small

glomeruli were only reliably visible if they were viewed

from the appropriate perspective, e.g., maG from the

medial surface. Previous studies (above references)

examined the distributions of glomeruli in sectioned ol-

factory bulbs and viewed their sections from a single

plane (e.g., horizontal), which apparently prevented the

detection of small glomeruli. We thus suggest that

Braubach et al.


future studies employ our technique to produce accu-

rate accounts of the distribution of glomeruli in zebra-

fish and related species.

Despite our improvements in labeling and detecting

glomeruli, we still observed regions that appeared to be

aglomerular. For example, the lateral plexus, which occu-

pies the posterolateral olfactory bulbs, appears to consist

of loosely organized axon terminals that only occasionally

aggregate in small, microglomerular structures (see also

Korsching, 2005; Meyerhof and Korsching, 2009), but do

not form anatomically distinct glomeruli as seen in other

areas of the olfactory bulb. Nevertheless, optophysiologi-

cal recordings show that certain amino acids elicit activity

in several small and discrete modules in the posterolat-

eral olfactory bulb (Friedrich and Korsching, 1997). Simi-

larly, both presynaptic terminals (pers. obs.) and mitral

cell dendrites (Friedrich and Laurent, 2001) form roughly

spherical synaptic tufts within the lP, suggesting that this

area may indeed contain organized glomerular structures.

Additional glomeruli may therefore be discovered in

future studies of the zebrafish olfactory system, espe-

cially if we have access to additional markers that enable

visualization of structural elements in the pre- and postsy-

naptic glomerular compartments.

Revision of the glomerular nomenclaturePrior to this study, nomenclatures for glomeruli had

been established in adult (Baier and Korsching, 1994)

and developing (Dynes and Ngai, 1998) zebrafish. Var-

iants of these nomenclatures were subsequently used to

describe glomeruli and/or olfactory bulb areas in anatom-

ical, physiological, and genetic studies of zebrafish olfac-

tion (Friedrich and Korsching, 1998; Sato et al., 2005;

Koide et al., 2009; Gayoso et al., 2011). Our results

required modifications of the original nomenclatures. For

example, we discovered new glomeruli in the anterodor-

sal olfactory bulbs and labeled these ‘‘dorsal glomeruli’’

(dG). This required us to change the names of the remain-

ing dorsal units, which were previously named dorsal- and

mediodorsal posterior glomeruli (Baier and Korsching,

1994). We thus renamed these glomeruli dlG and mdG,

respectively, in order to describe the glomeruli both by

their location in the olfactory bulbs and by their position

with reference to the newly identified glomeruli (see Table

2 for nomenclature comparisons). However, given that

several studies have already built on the previously estab-

lished nomenclatures, we applied new names only where

necessary.

Overall, we attempted to develop a revised nomencla-

ture that is simple, consistent, and accurate. Glomeruli

were thus given names based on: 1) their location on one

of the four olfactory bulb surfaces (e.g., dorsal), and 2)

their location in an anatomically distinct group (e.g., dor-

solateral). Some glomeruli could also be individually iden-

tified and were thus, 3) designated by a number (e.g., dor-

solateral glomerulus 1), or, in the case of anatomically

indistinguishable glomeruli, 4) designated by a letter

(e.g., dorsolateral glomeruli x). We believe that this

approach is simple and accurate, and that any glomerulus

that may in future be discovered through the use of new

and improved anatomical, functional, or molecular labels

can be assigned a name (i.e., a number) or a phenotype

(i.e., a letter) that is consistent with our nomenclature.

Neurochemistry of glomerular innervationWe used antibodies against G-protein a subunits and

calcium-binding proteins to identify neurochemically dis-

tinct OSNs and their innervation of glomeruli in different

olfactory bulb regions. Similar strategies have been

employed in zebrafish (Gayoso et al., 2011) and related

species (Frontini et al., 2003; Hansen et al., 2003), and

these data complement functional (Friedrich and Korsch-

ing, 1997, 1998), genetic (Sato et al., 2005; Koide et al.,

2009), and anatomical tracing studies (Hamdani et al.,

2001; Hamdani and Døving, 2002; Hansen et al., 2003)

to provide an overview of the organization and function of

the fish olfactory system.

Anti-Ga s/olf and anti-calretinin labelingA large population of OSNs labeled reliably with both

the anti-Ga s/olf and anti-calretinin antibodies. These

cells resembled the tall ciliated sensory OSNs that have

been described in zebrafish through genetic labeling

(Celik et al., 2002; Sato et al., 2005) and immunohisto-

chemistry (Castro et al., 2006; Gayoso et al., 2011). Cili-

ated OSNs have also been described in other species,

including catfish (Hansen et al., 2003), goldfish (Hansen

et al., 2004), and lampreys (Frontini et al., 2005). We

estimate that !100 glomeruli in ventromedial, dorsal,

and lateral olfactory bulbs are innervated by anti-Ga s/olf

and anti-calretinin labeled axons, and some of these glo-

meruli may thus receive innervation from ciliated OSNs.

Indeed, retrograde axon tracing from the ventromedial

zebrafish olfactory bulb labels ciliated OSNs (Gayoso

et al., 2011). Furthermore, Sato et al. (2005) used

genetic tracing to show that ciliated OSNs, which

express OR-type chemoreceptors, innervate the olfac-

tory bulb in an almost identical manner as we have

observed for anti-Ga s/olf and anti-calretinin labeled

axons. Finally, !102 OR receptor types appear to be

expressed in the zebrafish olfactory system (Hashiguchi

and Nishida, 2007), and the numbers of OR type chemo-

receptors correspond remarkably well with our esti-

mated number of anti-Ga s/olf and anti-calretinin labeled

glomeruli. We therefore hypothesize that anti-Ga s/olf

and anti-calretinin labeled zebrafish glomeruli mainly



encode activity from OR type receptors (green and ma-

genta shaded glomeruli Fig. 2).

Based on the combinatorial use of anti-Ga s/olf and

anti-calretinin antibodies we have furthermore provided

uniquely detailed insight into glomerular neurochemistry

in certain olfactory bulb regions. For example, we identi-

fied lG2 as a neurochemically distinct glomerulus in the

lateral olfactory bulb (see also Sato et al., 2005). Specif-

ically, lG2 was the only lG to label with the anti-Ga s/olf

antibody while all other lateral units labeled with anti-

calretinin. This observation implies that lG2 is innervated

by a distinct subset of OSNs that do not innervate other

lateral glomeruli. Indeed, Sato et al. (2005) demon-

strated that OR-type ciliated OSNs project to lG2; this is

distinct from the innervation of other lateral glomeruli,

which are innervated by microvillous OSNs. Further-

more, the selective labeling of lG2 suggests that this glo-

merulus may be innervated by a subset of ciliated

OSNs; these would be neurochemically distinct from

anti-Ga s/olf and anti-calretinin-labeled ciliated OSNs,

which innervate the majority of glomeruli (above). We

find multiple examples of selectively labeled glomeruli

throughout the olfactory bulbs and these are catalogued

in our olfactory system maps (Fig. 2).

Anti-calretinin labelingIn anti-calretinin-stained specimens, we commonly

detected cells in intermediate depths of the olfactory epi-

thelium. Morphologically, these cells resemble microvil-

lous cells that have been described in zebrafish (Sato

et al., 2005; Gayoso et al., 2011) and catfish (Hansen

et al., 2003). However, the calretinin IR OSNs were much

sparser in our specimens than microvillous cells identified

either through histochemical staining (Gayoso et al.,

2011) or genetic labeling (Sato et al., 2005) in zebrafish.

Furthermore, other groups have suggested that microvil-

lous cells may not express calretinin in zebrafish (Castro

et al., 2006; Gayoso et al., 2011). Other data, however,

indicate that OSNs with microvillous morphotypes stain

with anti-calretinin antibodies during zebrafish develop-

ment (Duggan et al., 2008). It is thus possible that a sub-

set of microvillous cells express calretinin and that their

detection is possible only during development or after tis-

sue processing, as described in our study.

We cannot confirm the detection of microvillous cells

based on anti-calretinin staining alone, but there exists

a correlation between calretinin IR axons and the

bulbar innervation by microvillous cells. Specifically, we

observed anti-calretinin-labeled axons in lateral and ven-

tromedial olfactory bulb regions. These regions contain

!18 lG, the lP and the vmG4–5, and correspond very well

with regions that are innervated by genetically labeled

microvillous OSNs (Sato et al., 2005). Glomeruli within

these regions (green glomeruli in Fig. 2) may therefore

encode the activity of V2R-type receptors that are

expressed by microvillous OSNs. Interestingly, there are

!46 V2R-type chemoreceptor genes in zebrafish (Hashi-

guchi and Nishida, 2007), but we could only identify 20

glomeruli proper in regions innervated by V2R-type micro-

villous cells. The activity of additional V2R OSN types

may therefore be encoded in the lP (see also Sato et al.,

2005). Indeed, the lP is selectively responsive to neutral

amino acids, while the anterolateral olfactory bulb, which

contains lG proper, responds preferentially to basic

amino acids (Friedrich and Korsching, 1997). Therefore,

V2R-type OSN activation appears to be encoded in de-

tectable glomeruli and within the fibrous lateral nerve

plexus.

Anti-S100 labelingThe morphologies of anti-S100-labeled OSN types are

consistent with previous descriptions of crypt and micro-

villous cells. Crypt cells have characteristic ovoid somata,

eccentric nuclei, and are located near the surface of the

olfactory epithelia of zebrafish (Germana et al., 2004;

Sato et al., 2005) and related species (Morita and Finger,

1998; Hansen et al., 2003). Furthermore, crypt cells

shown in previous studies appear to be sparse (Germana

et al., 2004), which is similar to our own observations.

Our results are thus consistent with previous reports.

We also identified OSNs with microvillous morpho-

types in anti-S100-stained epithelia, which is similar to

prior observations in zebrafish (Gayoso et al., 2011) and

related species (Hansen et al., 2003). However, there

are inconsistencies between our findings and those

reported earlier. For example, Gayoso et al. (2011)

observed widespread labeling of microvillous cells in

anti-S100-labeled tissue, whereas we observed only

very sparse anti-S100 labeling of OSNs. Similarly, Ger-

mana et al. (2004) failed to observe microvillous OSNs

in anti-S100-labeled tissue. To our knowledge, all of the

above-mentioned studies used antibodies from the same

supplier, but the fixation procedures employed in each

study were different. We therefore believe that inconsis-

tencies in OSN staining may arise from different fixation

methods used to prepare zebrafish olfactory epithelia for

immunohistochemical processing.

In the olfactory bulbs, we attained consistent labeling

of the large mdG2; this unit displayed anti-S100 labeling

in every specimen that we examined and we never

observed S100 labeling among other mdG. In contrast,

we occasionally observed widespread anti-S100 labeling

among the dG, dlG, and lG, but these observations were

inconsistent. Indeed, the widespread labeling of dorsal

glomeruli depicted in Figure 10A is more extensive than

anti-S100 labeling reported by Gayoso et al. (2011) and

Braubach et al.


also differs from results presented in Germana et al.

(2004), who did not report any anti-S100 labeled axons in

the olfactory bulbs. These inconsistencies, too, may be

due to different experimental approaches (see above),

and future examinations of anti-S100 expression in zebra-

fish olfactory bulbs will be helped by the development of

more reproducible staining techniques.

Other labels and remaining questionsOverall, our estimate on the number of glomeruli

(#140) correlates well with certain types of known zebra-

fish ORs (see above), but falls short of the total number of

predicted ORs (!167). Additional ORs include 18 trace

amine-associated receptor (TAAR) genes (Hashiguchi and

Nishida, 2007), the representation of which is unknown in

the olfactory system. These or other OR types might in-

nervate some of the eight large medial glomeruli for

which we could not resolve neurochemical details (gray

units in Fig. 2). These units cluster predominantly in the

medial olfactory bulbs, which have been linked to phero-

mone processing in several species (see also Hanson

et al., 1998; Hamdani and Døving, 2003, 2007), including

a single medial glomerulus in zebrafish (Friedrich and

Korsching, 1998). To our knowledge, however, the func-

tion of these glomeruli is unknown.

Anatomical variability of small butnot large glomeruli

We observed significant anatomical variability among the

small, anti-calretinin, and anti-Ga s/olf-labeled glomeruli in

the medial and dorsolateral olfactory bulbs. Throughout our

study we tried to minimize experimental bias or artifacts

that could underlie these variations by selecting only well-

stained preparations for analysis and confirming our find-

ings with multiple complementary labels. Yet some of the

perceived anatomical variability among small glomeruli

could have been due to observer errors. Specifically, the

axonal innervation was particularly dense in regions that

contained small glomeruli (e.g., vmGy), and glomeruli often

displayed overlapping innervation and unclear boundaries,

giving them a ‘‘fused’’ appearance. In contrast, large glo-

meruli were located in small groups and were clearly sepa-

rated from one another. Furthermore, axon bundles that in-

nervated large glomeruli were generally visible as soon as

they entered the olfactory bulbs, thus permitting their reli-

able and unambiguous detection.

Despite these issues, it is important to consider other

sources for the anatomical variations that we observed

among small glomeruli. Variations in glomerular numbers

are common in the animal kingdom, and do not appear to

depend solely on experimental techniques used to identify

glomeruli. For example, it has been reported that 20% of

glomeruli (in frogs, Nezlin and Schild, 2000; and moths,

Kazawa et al., 2009) to 10% of glomeruli (in bees, Flanagan

and Mercer, 1989; moths, Couton et al., 2009; Masante-

Roca et al., 2005; and ants Kleineidam et al., 2005) vary

between individuals. Similarly, the numbers and distribu-

tions of glomeruli also vary in the main (Royal and Key,

1999; Strotmann et al., 2000; Schaefer et al., 2001) and

accessory olfactory system of rodents (Meisami and Bhat-

nagar, 1998). The sources of such anatomical variation(s)

have not been determined, but they may be developmental.

There exists significant morphological variability during the

formation of glomeruli in mice (Royal and Key, 1999; Potter

et al., 2001) and sensory experience can influence their

maturation during postnatal development (Zheng et al.,

2000; Zou et al., 2004; Kerr and Belluscio, 2006; Oliva

et al., 2008). Thus, sensory experience could shape the as-

sembly of the zebrafish glomerular map in zebrafish, and

thereby produce variable phenotypes. In the absence of

detailed anatomical characterization of glomeruli, it has

been difficult to investigate such possibilities. The descrip-

tion of the zebrafish olfactory system presented by our and

previous studies should now make it possible to address

these important questions.

CONCLUSIONWe have provided the most comprehensive description

of the anatomical organization of the zebrafish olfactory

bulbs to date. Our data reveal that the zebrafish olfactory

bulbs contains diverse glomeruli, differing in size, neuro-

chemistry, and numbers. Furthermore, glomeruli can be

classified as being either individually identifiable or indis-

tinguishable from similar glomeruli within a cluster.

Future work can now build on the data that we present

here to examine in more detail the anatomy, function,

and development of these different types of glomeruli.

ACKNOWLEDGMENTSParts of this article were presented previously in the

doctoral dissertation of O.R.B. The SV2 antibody was

obtained from the Developmental Studies Hybridoma

Bank developed under the auspices of the NICHD and

maintained by the University of Iowa Biology Department

(Iowa City, IA). We thank Drs. Yoshihiro Yoshihara, Nobu-

hiko Miyasaka, and Tetsuya Koide for help with the glo-

merular nomenclature; Drs. Thomas Finger, Frank Smith,

and Richard Brown for criticism of earlier versions of the

article. We also thank Matt Stoyek and Dr. Russell Wyeth

for assistance with experiments, Annette Kolar, Dr. Kazue

Semba, and Dr. Yi-ling Hu for providing antibodies and

reagents, and Dr. David Hamilton for help with statistical

analyses. We also thank Dr. Lawrence Cohen for making

O.R.B. see red and permitting him to furiously edit this ar-

ticle while working at the Korea Institute of Science and

Technology and Yale University.



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