Ontogeny of the cocaine- and amphetamine-regulated transcript (CART) neuropeptide system in the brain of zebrafish, Danio rerio

Ontogeny of the Cocaine- and Amphetamine-Regulated Transcript (CART) NeuropeptideSystem in the Brain of Zebrafish, Danio rerio

Arghya Mukherjee, Nishikant K. Subhedar,* and Aurnab Ghose*

Indian Institute of Science Education and Research (IISER) Pune, Pashan, Pune 411021, India

ABSTRACTThe cocaine- and amphetamine-regulated transcript

(CART) peptidergic system is involved in processing

diverse neuronal functions in adult animals, including

energy metabolism. Although CART is widely distributed

in the brain of a range of vertebrates, the ontogeny of

this system has not been explored. The CART-immuno-

reactive system in the zebrafish central nervous system

(CNS) was studied across developmental stages until

adulthood. The peptide is expressed as early as 24

hours post fertilization and establishes itself in several

discrete areas of the brain and spinal cord as develop-

ment progresses. The trends in CART ontogeny suggest

that it may be involved in the establishment of commis-

sural tracts, typically expressing early but subsequently

decaying. CART elements are commonly overre-

presented in diverse sensory areas like the olfactory,

photic, and acoustico-mechanosensory systems,

perhaps indicating a role for the peptide in sensory

perception. Key neuroendocrine centers, like the pre-

optic area, hypothalamus, and pituitary, conspicuously

show CART innervations, suggesting functions analo-

gous to those demonstrated in other chordates.

Uniquely, the epiphysis also appears to employ CART

as a neurotransmitter. The entopeduncular nucleus is a

major CART-containing group in the adult teleost fore-

brain that may participate in glucose sensing. This

region responds to glucose in the 15-day larvae, sug-

gesting that the energy status sensing CART circuits is

active early in development. The pattern of CART

expression in zebrafish suggests conserved evolutionary

trends among vertebrate species. Developmental

expression profiling reveals putative novel functions and

establishes zebrafish as a model to investigate CART

function in physiology and development. J. Comp.

Neurol. 520:770–797, 2012.

VC 2011 Wiley Periodicals, Inc.

INDEXING TERMS: teleost; developmental expression profiling; energy metabolism

In recent years cocaine- and amphetamine-regulated

transcript (CART) has emerged as one of the most widely

distributed peptides in the brain of vertebrates (Koylu

et al., 1998; Lazar et al., 2004; Singru et al., 2007), and

its physiological significance has been intensely investi-

gated. The role of CART in the regulation of food intake

in mammals is well established. CART mRNA is

expressed in the arcuate and paraventricular nuclei that

regulate feeding (Douglass et al., 1995; Kristensen

et al., 1998), and the peptide is involved in the process-

ing of appetite-related information (Vrang et al., 1999,

2000). Although the administration of CART inhibits

feeding in a dose-dependent manner (Lambert et al.,

1998; Kuhar and Dall Vechia, 1999; Thim et al., 1999;

Larsen et al., 2000), CART mRNA expression is reduced

in fasting conditions (Kristensen et al., 1998; Ahima

et al., 1999).

In addition to its role in energy metabolism, the peptide

is involved in a range of activities like neuroendocrine reg-

ulation (Stanley et al., 2001; Baranowska et al., 2003;

Larsen et al., 2003; Baranowska et al., 2004; Wittmann

et al., 2005), learning and memory (Upadhya et al.,

2011), and affective disorders like anxiety and depression

Grant sponsor: Indian Institute of Science Education and Research(IISER) Pune; the Department of Science and Technology (DST),Government of India; Grant number: SR/SO/AS-06/2010.

*CORRESPONDENCE TO: Aurnab Ghose and Nishikant K. Subhedar,Indian Institute of Science Education and Research (IISER) Pune, SaiTrinity Building, Garware Circle, Pashan, Pune 411021, India. E-mail:[email protected] and [email protected]

VC 2011 Wiley Periodicals, Inc.

Received May 4, 2011; Revised July 25, 2011; Accepted September 9,2011

DOI 10.1002/cne.22779

Published online October 18, 2011 in Wiley Online Library(wileyonlinelibrary.com)

770 The Journal of Comparative Neurology |Research in Systems Neuroscience 520:770–797 (2012)

RESEARCH ARTICLE

(Dandekar et al., 2008, 2009). It also influences locomo-

tor activity (Kimmel et al., 2000) and reward mechanisms

(Jaworski et al., 2003a,b), and interacts with the endoge-

nous opioid system (Damaj et al., 2004). In teleosts, the

role of CART as an anorexic agent is well documented

(Kristensen et al., 1998; Volkoff and Peter, 2000; Kobaya-

shi et al., 2008). Recently, the involvement of the peptide

in the neuroendocrine regulation of reproduction and in

energy metabolism was demonstrated in catfish (Barsa-

gade et al., 2010; Subhedar et al., 2011).

However, information on the ontogeny of CART in the

vertebrate brain is not available. Zebrafish, Danio rerio, is

an important model system for genetic and developmen-

tal investigations of the vertebrate central nervous sys-

tem (CNS) (Kimmel, 1993; Westerfield, 1993; Rupp et al.,

1996; Wullimann et al., 1996). It is amenable to genetic

and pharmacological manipulations, neuronal ablations

(Abraham et al., 2010), and optogenetic regulation (Wyart

et al., 2009) and is widely employed in behavioral and

cognitive studies (Guo, 2004; Linney et al., 2004). The

present study examines the developmental expression

patterns of CART containing elements in the brain, retina,

and spinal cord of the zebrafish across larval, juvenile,

and adult stages.

Recent studies reveal that the CART-containing neu-

rons in the telencephalon of the catfish are sensitive to

glucose and may play a role in energy metabolism (Sub-

hedar et al., 2011). With a view to evaluating how early in

development analogous functional circuits appear in the

brain of developing zebrafish, the response of CART ele-

ments to glucose treatment at the 15-day post fertiliza-

tion (dpf) stage was assessed. The present study not only

Abbreviations

A anterior thalamic nucleusAC anterior commissureBC brachium conjunctivumC central canalCART cocaine- and amphetamine-regulated transcriptCC corpus cerebelliCP central posterior thalamic nucleusCv commissura ventralis rhombencephaliD dorsal telencephalic areaDc central zone of the dorsal telencephalic areaDd dorsal zone of the dorsal telencephalic areaDH dorsal hornDiV diencephalic ventricleDIL diffuse nucleus of the inferior hypothalamic lobeDl lateral zone of the dorsal telencephalic areaDm medial zone of the dorsal telencephalic areadpf days post fertilizationDP dorsal posterior thalamic nucleusDp posterior zone of the dorsal telencephalic areaDT dorsal thalamusDTN dorsal tegmental nucleusE epiphysis/pineal glandECL external cell layer of the olfactory bulbEN entopeduncular nucleusEG eminentia granularisEW Edinger-Westphal nucleusEy eyeFd funiculus dorsalisFld funiculus lateralis (pars dorsalis)Flv funiculus lateralis (pars ventralis)FP floor plateFv funiculus ventralisGCL ganglion cell layerGL glomerular layer of the olfactory bulbHa habenulaHaC habenular commissureHC horizontal commissureHc caudal zone of periventricular hypothalamusHi intermediate zone of periventricular hypothalamushpf hours post fertilizationHr rostral zone of periventricular hypothalamusHv ventral zone of periventricular hypothalamusHy hypophysis/pituitary glandICL internal cell layer of olfactory bulbIMRF intermediate reticular formationIPL inner plexiform layerIO inferior oliveIRF inferior reticular formationL lensLCa lobus caudalis cerebelliLH lateral hypothalamusLLF lateral longitudinal fascicleLVII lobus facialisLR lateral recess of diencephalic ventricle

LRN lateral reticular nucleusLX lobus vagusM1 migrated posterior tubercular area

(future preglomerular complex)MLF medial longitudinal fascicleMO medulla oblongataN nucleus of the medial longitudinal fascicleNPY neuropeptide YNT neural tubeOB olfactory bulbOC optic chiasmaOE olfactory epithelium/placodeON optic nerveOTr optic tractP palliumPC posterior commissurePG preglomerular nucleusPGZ periventricular gray zone of optic tectumPM magnocellular preoptic nucleusPOA preoptic areapoc postoptic commissurePPa anterior part of parvocellular preoptic nucleusPPp posterior part of the parvocellular preoptic nucleusRL rhombic lipRV rhombencephalic ventricleS subpalliumSAC stratum album centraleSC spinal cordSCN suprachiasmatic nucleusSFGS stratum fibrosum et griseum superficialeSO stratum opticumSM stratum marginaleSR superior rapheSRF superior reticular formationSV saccus vasculosusTeO tectum opticumTeV telencephalic ventricleTL torus longitudinalisTLa torus lateralisTPp periventricular nucleus of posterior tuberculumTS torus semicircularisV ventral telencephalic areaVal lateral division of valvula cerebelliVam medial division of valvula cerebelliVC valvula cerebelliVc central nucleus of the ventral telencephalic areavcc ventrocaudal clusterVd dorsal nucleus of the ventral telencephalic areaVH ventral hornVl lateral nucleus of the ventral telencephalic areaVM ventromedial thalamic nucleusVs supracommissural nucleus of the ventral telencephalic areaVT ventral thalamusVv ventral nucleus of the ventral telencephalic area

CART expression during zebrafish development

The Journal of Comparative Neurology |Research in Systems Neuroscience 771

provides insight into the neuroanatomy of the CART sys-

tem in the developing vertebrate CNS, but may also point

to novel developmental functions, physiological rele-

vance, and evolutionary trends.

MATERIALS AND METHODS

Animal handling and sampling proceduresAll experimental procedures were performed according

to the guidelines of the Institutional Animal Ethics Com-

mittee (IAEC) of IISER, Pune, under the Committee for the

Purpose of Control and Supervision of Experiments for

Animals (CPCSEA), New Delhi, India. All experiments con-

formed to international guidelines on the ethical use of

animals. Wild-type Indian strain zebrafish (Danio rerio) of

the long fin variety were used in the present study. The

fish were housed in multiplexed recirculating tanks

(Aquatic Habitats, Beverly, MA) and maintained at

28.5�C. The system was supplied with de-hardened water

and treated with activated charcoal and ultraviolet radia-

tion. Regular water quality checks were performed to

maintain optimum water quality (hardness 100–300 mg/

L of CaCO3; alkalinity 50–300 mg/L of CaCO3; nitrate <

20 mg/ml; pH 6–8; conductivity 180–350 lS).Male and female zebrafish were mated according to a

standard protocol, and the fertilized eggs were collected.

Embryos were maintained in an incubator at 28.5�C in E3

medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33

mM MgSO4, 0.00001% [w/v] methylene blue) until 7 or

15 dpf, following which they were transferred to the recir-

culating tank. Developing zebrafish at 24 and 48 hours, at

4, 7, 15, and 30 days, and at 6 months post fertilization

stages were sampled for immunohistochemical analysis.

At least five animals were used for each stage. Until 15

dpf, the fish were anesthetized in 2-phenoxy ethanol

(1:2,000), immersed in Bouin’s fixative, and stored at 4�Cfor 18 hours. For later stages, the fish were anesthetized,

and the brains were surgically exposed from the dorsal

side and immersed in fixative. The brains were dissected,

cryoprotected overnight in 25% sucrose in 1X phosphate-

buffered saline (PBS, pH 7.4), and cryosectioned at

10-lm thickness on a cryostat (Leica CM1850, Leica

Microsystems, Nussloch, Germany) at �18�C in sagittal

or transverse planes.

ImmunofluorescenceThe tissue sections were processed for immunocyto-

chemical labeling according to standard immunofluores-

cence protocol. Briefly, the tissue sections were rinsed in

1X PBS containing 0.1% Triton X-100 (washing solution)

for 15 minutes, followed by treatment with 5% normal

goat serum in PBS containing 0.1% Triton X-100 (blocking

solution) for 1 hour. The sections were incubated with

monoclonal antibodies against CART (55–102) (Thim

et al., 1999) or polyclonal anti-neuropeptide Y (NPY) anti-

body (N9528; Sigma, St. Louis, MO). The antibodies were

diluted in blocking solution (CART, 1:2,000; NPY, 1:5,000;

see Table 1), and sections were incubated overnight in a

humid atmosphere at 4�C. The sections were rinsed in

washing solution, and then treated with blocking solution

for 30 minutes. Secondary antibody incubations were car-

ried out with anti-mouse or anti-rabbit IgG-conjugated

Alexa Fluor 488 or 568 (A-11001; A-11004; A-11008; A-

11011; Invitrogen, Carlsbad, CA) in a humid chamber for

3 hours at room temperature. The sections were mounted

in a glycerol-based mounting medium (90% glycerol, 0.5%

N-propyl-gallate, 20 mM Tris, pH 8.0) containing 40,6-dia-midino-2-phenylindole (DAPI; 1 lg/ml) and observed

under an epifluorescence microscope (AxioImager Z1,

Carl Zeiss, Gottingen, Germany). The desired fields from

the sections were photographed by using a CCD camera

(AxioCam MRm, Carl Zeiss), and the images were

adjusted for size, contrast, and brightness by using Adobe

Photoshop (San Jose, CA). Adobe Illustrator was used to

prepare the panels and diagrammatic representations.

For whole-mount immunofluorescence, 24-hour larvae

were fixed in 4% paraformaldehyde overnight at 4�C. Sub-sequently they were rinsed three times with 1X PBS with

1% Triton X-100 (washing solution) and placed in blocking

solution (10% normal goat serum in washing solution) for

1 hour. The larvae were then incubated in monoclonal

antibodies against CART (55–102) diluted in blocking so-

lution (1:1,000) overnight at 4�C. The larvae were then

TABLE 1.

Primary Antibodies Used

Antigen Immunogen Source Dilution

Cocaine- and amphetamine-regulated transcript (CART)

Recombinant rat CART(aa 55–102)

Mouse monoclonal antibodiesgenerated by Lars Thim andJes Clausen, Novo Nordisk,Denmark (Thim et al., 1998)

1:2,000 for cryosections1:1,000 for whole mounts

Neuropeptide Y (NPY) Synthetic peptide based onfull-length porcine NPY

(GeneID 397304)conjugated to KLH

Rabbit polyclonal; Sigma,St. Louis, MO, cat.

# N9528

1:5,000

Mukherjee et al.

772 The Journal of Comparative Neurology |Research in Systems Neuroscience

Figure 1. A: CLUSTAL W alignment of CART peptide sequence of R. norvegicus (P49192), D. rerio I (NP_00101757.1), and D. rerio II

(XP_685429.2). The consensus across the sequences is shown with histograms. A black bar on top depicts the region against which the

CART antibody was raised. B: Positive control showing CART immunoreaction, with antibodies against CART, in the cells (arrows) and fibers

(arrowheads) of the entopeduncular nucleus (EN) of zebrafish. C: Negative control showing no immunoreactivity of EN after incubation

with antibodies preadsorbed with CART. D: Positive control showing NPY immunoreaction, with antibodies against NPY, in the cells

(arrows) and fibers (arrowheads) of the entopeduncular nucleus (EN) of zebrafish. E: Negative control showing no immunoreactivity in the

EN after incubation with antibodies preadsorbed with NPY. Scale bar ¼ 20 lm in A–E.



TABLE2.

DistributionofCARTIm

munoreactivityin

theBrain

andSpinalCord

oftheDevelopmentalStagesofZebrafish

1

6m

(adult)

30dpf

15dpf

7dpf

4dpf

48hpf

Olfactory

bulb

F:GL,scatteredin

OB

F:GL,scatteredin

OB

F:GL

F:GL

F:GL

F:presumptive

GL

Telencephalon

F:Dc,

Dd,Vs,

Vl,

Vv,

Vd,ACC:EN,

Vv,Vd

F:Dc,

Dd,Vs,

Vl,

Vv,Vd,ACC:EN

F:D,V,ACC:EN

F:D,V,ACC:EN

F:P,AC

F:P,AC

Epithalamus

F:E,Ha,HaC

F:E,Ha,HaC

F:E,Ha,HaCC:E

F:E,Ha,HaCC:E

F:E,Ha,HaC

F:Ha,HaC/PC

Thalamus

F:A,VM,TPp,PC,

CP,PGC:VM,CP

F:TPp,PC,CP,PG

C:CP

F:VT,

DT,

PC,CP

C:CP

F:VT,

DT,

PC,CP

C:CP

F:DT,

VT,

PCC:CP

F:DT,

VTC:DT

Hypothalamus

F:POA,PM,PPp,

SCN,poc,

HC,Hv,

Hc,

LH,DIL,SV,Hy

C:Hc

F:POA,PM,PPp,

SCN,poc,

HC,Hv,

Hc,

LH,DIL,Hy

C:Hc

F:POA,poc,

Hr,Hi,

Hc,

LH,DIL,Hy

F:POA,poc,

Hr,Hi,

Hc,

LH,DIL

F:presumptive

POA,

poc,

Hr/Hi,LH

F:poc

Optictectum

F:SOþ

SM,SFGS,

SACC:PGZ

F:SOþ

SM,SFGS,

SACC:PGZ

F:SOþ

SM,SFGS,

SAC

F:SOþ

SM,SFGS,

SAC

F:SOþ

SM,SAC

Nil

Pretectum/Tegmental

areas

F:DTN

,TL,TS

,TLa,

EW

C:DTN

,TS

,EW

F:DTN

,TL,TS

,TLa

F:TL,TS

F:TL,TS

F:presumptive

pretectum

F:presumptive

pretectum

Synencephalon

F:NC:N

F:NC:N

F:NC:N

F:NC:N

F:NC:N

n.a.

Cerebellum

F:VC,Val,Vam,

CC,LCa,EG

F:VC,Vam,CC,

LCa,EG

F:CC

F:CC

Nil

n.a.

Facialandvagal

lobes

F:LVII,LXC:LVII

F:LVII,LXC:LVII

n.a.

n.a.

n.a.

n.a.

Medulla

oblongata

F:SRF,IO,IRF,

LRN,IM

RF,SR,Fv,

Flv,Fld

C:BC,SR

F:SRF,IO,IRF,

LRN,IM

RF,SR,Fv,

Flv,Fld

C:BC,SR

F:SRF,IO,IRF,

LRN,IM

RF,SR

C:MO,SR

F:SRF,SR,IO,

IMRF,IRFC:MO

F:ventrolateral

C:MO

F:ventolateral

C:MO

Spinalcord

F:DH,VH,Fd,Fld

Flv,Fv

F:DH,VH,Fd,Fld

Flv,Fv

F:SC

F:SC

F:SC

F:SC

Retina

F:GCL,IPL,OC,OTr

C:IPL

F:GCL,IPL,OC,OTr

C:IPL

F:GCL,IPLC:IPL

F:GCL,IPLC:IPL

F:GCL,IPLC:IPL

Nil

Abbreviations:

CART,

cocaine-andamphetamine-regulatedtranscript;F,fibers;C,cells;n.a.,notapplicable

(indicatingthattheregionis

notdelineatedatthis

stage);Nil,

lack

ofCARTelements.Forother

abbreviations,

seelist.

1The24-hpfstageisnotincludedin

Table

2.Atthisstage,CART-positive

cells

andfibers

are

seenin

thecaudalsectionsofthebrain

correspondingto

thevccanditsprojections(Fig.2).

Mukherjee et al.


rinsed with washing solution for 2 hours with three

changes. Secondary incubation was carried out with anti-

mouse IgG conjugated to Alexa Fluor 488 overnight at

4�C with agitation. The larvae were mounted on a drop of

1% low melting agarose on a coverslip and observed

under an inverted laser scanning microscope (LSM 710,

Carl Zeiss). Images were adjusted for size, contrast, and

brightness by using Adobe Photoshop.

Neuroanatomical areas in the zebrafish brain, at different

stages of development, were identified on the basis of ear-

lier descriptions (Wullimann et al., 1996; Mueller and Wulli-

mann, 2005; Castro et al., 2009) and DAPI counterstaining.

Antibody characterizationThe monoclonal primary antibodies against CART (55–

102) used in the present study were prepared (Thim

et al., 1999) and given to us by Drs. Lars Thim and Jes

Clausen (Novo Nordisk, Bagsvaerd, Denmark). The anti-

bodies were originally characterized for specificity in the

rat brain. CLUSTAL W alignment (Thompson et al., 1994)

of rat CART (P49192) with the two predicted CART

sequences from D. rerio, NP_001017570.1 and

XP_685429.2 (Kobayashi et al., 2008), indicated a high

degree of identity, particularly in the region of CART used

to generate the antibodies (Fig. 1A).

Recently these antibodies have been shown to react

specifically with the CART peptide in the brain of the tele-

osts Clarias batrachus and C. gariepinus (Singru et al.,

2007; Barsagade et al., 2010; Subhedar et al., 2011).

In these studies, preadsorption with the peptide

(54–102) was used to demonstrate specificity. Similarly,

various control procedures were conducted to test the

possibility of cross-reactivity in the zebrafish brain. Ento-

peduncular nucleus (EN)-containing sections were incu-

bated with diluted antibodies preadsorbed with CART

peptide (54–102; a generous gift from Drs. Lars Thim and

Jes Clausen, Novo Nordisk) at 10�5 M for 24 hours prior

to incubation. The procedure resulted in total loss of the

immunoreactivity from the sections, confirming the speci-

ficity of the antibodies (Fig. 1B,C).

To immunolabel NPY elements, antibodies raised

against porcine NPY (Sigma; details provided in Table 1)

were employed. These antibodies have been extensively

Figure 2

Figure 2. Demonstration of CART immunoreactivity in the neural

tube of zebrafish at 24 hpf. Drawing in upper left shows the level

of the transverse section represented in A. A: Schematic drawing

of transverse section showing CART neurons in the ventrocaudal

cluster of the neural tube (dark circles) and fibers (dots). The an-

atomical areas are indicated on the left, and the CART cells and

fibers are shown on the right in this and the rest of the sche-

matic representations. B: CART-immunoreactive cells (arrows) and

fibers (arrowheads) can be seen in the neural tube (NT) at the

24-hpf stage. C: A maximum intensity projection of a confocal

stack of a paradorsally oriented whole-mount 24-hpf embryo im-

munostained with CART antibodies. Rostral is oriented to the left.

Immunoreactive cells in vcc (arrows) and fibers (arrowheads) aris-

ing from the vcc are marked. For abbreviations, see list. Scale

bar ¼ 200 lm in drawing, upper left; 30 lm in A,B; 50 lm in C.



Figure 3. CART immunoreactivity in the developing CNS and eye of zebrafish at 48 hpf. Drawing at the upper left shows a lateral view of

the brain indicating the levels (A–E) represented in the corresponding transverse sections showing CART-immunoreactive cells (dark

circles) and fibers (dots). F–I: Transverse sections through a 48-hpf larva showing CART immunoreactivity in the cells (arrows) and fibers

(arrowheads) of presumptive olfactory bulb (OB) and pallium (P), anterior commissure (AC) and habenular commissure/posterior commis-

sure (HaC/PC), dorsal thalamus (DT), ventral thalamus (VT), postoptic commissure (poc), and medulla oblongata (MO). G and H are mon-

tages of photomicrographs taken from adjacent sections. J: Sagittal section of the brain shows CART immunoreactivity traceable from the

thalamic area (DT) through the anterior commissure (AC) as far as the presumptive olfactory bulb (OB). H,H0: Thalamus cells (H), with

higher magnification view (H0) from the framed area in H. I,I0: MO cells (I), with magnified view (I0)of the framed area. For abbreviations,

see list. Scale bar ¼ 100 lm in drawing, upper left; 50 lm in E (applies to A–E and F–J).

Mukherjee et al.


used to label the NPY cells and fibers in the brain of cat-

fish (Gaikwad et al., 2004) and tilapia (Sakharkar et al.,

2005). Preadsorption of the antibodies with synthetic

human peptide (Sigma, N5017) eliminated the immunore-

action in these as well as the present study (Fig. 1D,E).

Omission of the primary antibodies, CART as well as

NPY, from incubation medium also eliminated the

immunoreaction.

Glucose treatment of zebrafish larvaeFirst, 15 dpf zebrafish were immersed in 2% glucose dis-

solved in E3 medium for 3 hours and their brains were

Figure 4. Schematic drawings of CART immunoreactivity in the CNS and eye of zebrafish at 4 dpf. Drawing at the upper left shows lateral

view of the brain indicating the levels (A–G) represented in the corresponding transverse sections showing CART-immunoreactive cells

(dark circles) and fibers (dots). For abbreviations, see list. Scale bar ¼ 200 lm in drawing, upper left; 50 lm in F (applies to A–G).



processed for immunofluorescence. Control fish were

immersed in E3 medium without glucose. The response

was evaluated in terms of the change in number of CART-

positive cells in the EN. Serial sections of the telencepha-

lon containing EN (bilaterally; six serial sections) from

glucose-treated and control brains (n ¼ 6 for each group)

were sampled. For photomicrography, the exposure time

was held constant, and DAPI fluorescence was used to

normalize across sections in some cases. The CART-

immunofluorescent cells of the EN were manually counted.

Immunofluorescent cells with a clearly demarcated neu-

rite, typical signal enrichment at the neurite base, and a

distinct DAPI stained nuclei were scored. A total of 36

sections from each group was examined and expressed

as number of cells per section. To avoid overestimation,

the data were processed according to Abercrombie’s

method (Abercrombie, 1946) by using the equation, N ¼(n � D)/(D þ T), where N ¼ number of neurons, n ¼ nu-

clear profile counts, D ¼ mean nuclear diameter, and T ¼section thickness. The data were statistically analyzed by

using two-tailed, unpaired Student’s t-test.

RESULTS

The various areas and neuronal groups in the brain of

developing zebrafish were identified on the basis of the

cytoarchitectonic features described in earlier studies

(Wullimann et al., 1996; Mueller and Wullimann, 2005;

Castro et al., 2009) and DAPI staining. The data on the

ontogeny of CART expression in the brain of larva, juve-

nile and adult zebrafish are summarized in Table 2 and

Figs. 2–15.

Figure 5. Rostrocaudal series of transverse sections (A–I) of the CNS and eye of zebrafish at 4 dpf showing CART immunoreactivity

in the cells (arrows) and fibers (arrowheads) of olfactory glomeruli (GL; A) and anterior commissure (AC; B). Fibers are seen in the

pineal gland (E; C) and in the posterior commissure (PC; D). E: Immunoreactive fibers are distributed in the stratum album centrale

(SAC) of the optic tectum, dorsal thalamus (DT), ventral thalamus (VT), and postoptic commissure (poc). F: In the eye, immunoreac-

tive amacrine cells are seen in the inner plexiform layer (IPL), and fibers are seen in the ganglion cell layer (GCL). G–I: In caudal

areas, CART-positive cells and scattered fibers are seen in the central posterior thalamic nucleus (CP; G), nucleus of the medial lon-

gitudinal fasciculus (N; H), and medulla oblongata (MO; I). J: Sagittal section of the brain showing CART-positive cells (boxed area is

magnified in the inset), and fibers traceable from the MO to the spinal cord (SC). For abbreviations, see list. Scale bar ¼ 50 lm in

A–E,J; 20 lm in F–I.

Mukherjee et al.


CART in early developmentIn transverse sections of the 24-hpf larvae, a small

cluster of neurons was located laterally in the rostral NT;

a few fibers were also seen in the vicinity (Fig. 2A,B).

Whole-mount confocal analysis of CART antibody-stained

24-hpf larva revealed that this bilaterally located group

Figure 6. Schematic drawings of CART immunoreactivity in the CNS and eye of zebrafish at 7 dpf. Drawing at the upper left shows lateral

view of the brain indicating the levels (A–J) represented in the corresponding transverse sections showing CART-immunoreactive cells

(dark circles) and fibers (dots). K: Schematic representation of a section of the eye showing CART immunofluorescence. For abbreviations,

see list. Scale bar ¼ 200 lm in drawing, upper left; 45 lm in K (applies to A–K).



corresponds to the ventrocaudal cluster (vcc) in the mid-

brain (Fig. 2C). These neurons issue CART-positive proc-

esses that extend caudally and contribute prominently

to the medial longitudinal fascicle (MLF). Rostrally, the

vcc is reported to send fibers to a number tracts includ-

ing the postoptic commissure (poc), the tract of the

postoptic commissure, and the posterior commissure

(PC) (Chitnis and Kuwada, 1990). Our study suggests

that these tracts may contain some CART-ergic fibers

(Fig. 2C).

At 48 hpf, immunoreactive fibers were seen in the

developing olfactory bulb (OB), but not in the olfactory

epithelial placode (OE; Fig. 3A,F). Immunostained fibers

were seen in the pallium (P), anterior commissure (AC),

poc, and presumptive habenular/posterior commissure

(HaC/PC; Fig. 3). In addition to a cluster of three to four

Figure 7. Rostrocaudal series of transverse sections (A–G) of CNS and eye of zebrafish at 7 dpf showing CART-immunoreactive cells

(arrows) and fibers (arrowheads). Dense immunoreactivity is seen in the olfactory glomeruli (GL; A) and dorsal (D) and ventral telencepha-

lon (V; B). C,C0,D: CART-positive fibers (C) and cells (C0) are seen in the entopeduncular nucleus (EN) and also in the pineal gland (E; D).

Strong immunofluorescence is seen in the anterior commissure (AC; C). E: Intensely stained fibers are seen in the preoptic area (POA)

and postoptic commissure (poc). F: Intensely stained fibers pass through the posterior commissure (PC) and bilaterally connect the

migrated posterior tubercular area (M1). Immunoreactive fibers are also seen in the inner plexiform layer (IPL) and the ganglion cell layer

(GCL). G: Few CART-positive cells of the central posterior thalamic nucleus (CP) are seen along with dot like fibers in the dorsal thalamus

(DT). For abbreviations, see list. Scale bars ¼ 50 lm in A–C,E,F; 20 lm in C0,D,G.

Mukherjee et al.


immunoreactive cells in the dorsal thalamus (DT), CART

fibers were seen in the dorsal as well as ventral thalamus

(VT; Fig. 3C,H,H0). In the sagittal sections, these cells

appear to issue fibers that extend rostroventrally, probably

decussating in the AC and/or in the poc and finally termi-

nating in the OB area (Fig. 3J). A few immunoreactive cells

and a band of fibers were seen in the ventrolateral medulla

oblongata (MO; Fig. 3D,I,I0). These fibers, probably emerg-

ing from the CART-positive cells, were traceable through-

out the spinal cord (SC; Fig. 3E). No CART perikarya were

detected in the SC at any developmental stage.

From 4 dpf onward, major areas like the OB, telen-

cephalon, thalamus, hypothalamus, optic tectum (TeO),

cerebellum, MO, and SC were clearly demarcated. The

organization of CART-containing systems in each lobe, at

each stage of development, is described in the following

sections.

CART in the olfactory systemIntensely stained CART fibers were seen in the olfac-

tory glomeruli of the zebrafish from 4 dpf through adult-

hood (6 months); indeed, the CART immunoreactivity

demarcates the profile of each glomerulus (Figs. 4–7A,

9–13A). Fine dot-like immunoreactivity was seen in the

granular layer of the bulb of the 30 dpf and older fish

(Figs. 11A, 12A, 13A). In addition, isolated varicose fibers

Figure 8. Rostrocaudal series of transverse sections (A–E) of CNS and eye of zebrafish at 7 dpf showing CART-immunoreactive cells

(arrows) and fibers (arrowheads). A: The optic tectum shows beaded fibers in the stratum opticum (SO), stratum marginale (SM), and stra-

tum album centrale (SAC). A network of fibers is seen in the DT and ventral thalamus (VT), which extends ventrally as far as the caudal

zone of periventricular hypothalamus (Hc). B: Intense immunoreactivity is seen in the cells of the nucleus of the medial longitudinal fasci-

culus (N). C: Dense fibers are seen in the ventral part of the medulla oblongata and the caudal zone of periventricular hypothalamus (Hc).

D: The caudal medulla oblongata (MO) shows a few immunoreactive cells and scattered fibers. E: The spinal cord (SC) shows lateral distri-

bution of CART-positive fibers. For abbreviations, see list. Scale bars ¼ 50 lm in A,C,E; 20 lm in B,D.



Figure 9. Schematic drawings of CART immunoreactivity in the CNS and eye of zebrafish at 15 dpf. Drawing at upper left shows lateral

view of the brain indicating the levels (A–L) represented in the corresponding transverse sections showing CART-immunoreactive cells

(dark circles) and fibers (dots). M: Schematic representation of a section of the eye showing CART immunofluorescence. For abbreviations,

see list. Scale bar ¼ 200 lm in drawing, top left; 100 lm in M (applies to A–M).

Mukherjee et al.


were seen throughout the bulb at these stages. No CART

immunoreactivity was detected in the olfactory organ at

any stage of development.

CART in the telencephalon and preoptic areaIn the 4 dpf larva, CART-immunoreactive fibers in the P

seemed to descend and decussate in the AC (Figs. 4B,

5B). These could be traced in the lateral thalamus, and

further caudally as far as the neurons of the nucleus of

the medial longitudinal fasciculus (N; Figs. 4E, 5H). In the

7 dpf larva, whereas the rostral telencephalon showed a

dense network of CART fibers, few neurons in the pre-

sumptive EN showed intense immunofluorescence

(Figs. 6B,C, 7B,C,C0). Intensely stained fibers were also

seen in the AC and preoptic area (POA; Figs. 6C,D, 7C,E).

Figure 10. Rostrocaudal series of transverse sections (A–L) of CNS and eye of zebrafish at 15 dpf showing CART-immunoreactive cells

(arrows) and fibers (arrowheads). A: Immunoreactivity is seen in the olfactory glomeruli (GL) and the dorsal telencephalic area (D). B: In the

eye, CART-positive amacrine cells are seen in the inner plexiform layer (IPL). C: Dense immunoreactivity is seen in the preoptic area (POA),

whereas the optic nerve (ON) shows stray fibers with moderate intensity. Immunoreactive fibers are also seen in the IPL and the ganglion

cell layer (GCL). D: The entopeduncular nucleus (EN) shows a few CART-positive cells. E: Dense fibers are seen in the anterior commissure

(AC) and POA. F: Beaded fibers are seen in the posterior commissure (PC) and preglomerular area (PG). G: Scattered fibers are seen in the

caudal zone of periventricular hypothalamus (Hc) and the pituitary gland (Hy). H: The pineal gland (E) shows intensely stained cells as well

as fibers. I: A few CART-positive cells of the central posterior thalamic nucleus (CP) are seen along with dot-like fibers in the dorsal thalamus

(DT). J,K: In caudal areas, CART-positive cells and scattered fibers are seen in the nucleus of the medial longitudinal fasciculus (N; J) and

superior raphe (SR; K). The superior reticular formation (SRF; K) also shows scattered CART fibers. L: The lateral spinal cord (SC) shows sev-

eral intense CART-positive fibers. For abbreviations, see list. Scale bars ¼ 50 lm in A,C,E–G,L; 20 lm in B,D,H–J,K.



In the caudal sections, a narrow patch of intensely

stained fibers was seen in the dorsal telencephalon, on

either side of the pineal gland (E; Fig. 6D).

CART-immunoreactive cells were prominently seen in

the EN of the telencephalon from the 15 dpf stage

through adulthood (Figs. 9C,D, 10D, 11C,D, 12C,

13C,D,E, 14D). In the 15 dpf larva, fibers were seen in

dorsal and ventral parts of the telencephalon (Figs.

9B,C). In the dorsal telencephalon of the 30 dpf juve-

niles, dense CART immunoreactity was seen in the

Figure 11. Schematic drawings of CART immunoreactivity in the CNS of zebrafish at 30 dpf. Drawing at upper left shows lateral view of

the brain indicating the levels (A–N) represented in the corresponding transverse sections showing CART-immunoreactive cells (dark

circles) and fibers (dots). For abbreviations, see list. Scale bar ¼ 200 lm in drawing, upper left; 50 lm in M (applies to A–N).

Mukherjee et al.


fibers of the dorsal (Dd) and central zones (Dc; Figs.

11C,D, 12B). However, at this stage, the medial (Dm),

lateral (Dl), and posterior zones (Dp) were mostly devoid

of immunoreactivity. As in the previous stages, the 15

dpf larva showed several CART fibers in the AC (Figs.

9D,E, 10E). Although these seemed to reduce with

Figure 12. Rostrocaudal series of transverse sections (A–P) of CNS of zebrafish at 30 dpf showing CART-immunoreactive cells (arrows)

and fibers (arrowheads). A: Olfactory glomeruli (GL) showing immunoreactivity. B: CART-positive fiber distribution in the various parts of

the dorsal telencephalon: dorsal zone (Dd), central zone (Dc). The medial zone (Dm), lateral zone (Dl), and posterior zone (Dp) do not

show CART fibers. C: Immunoreactive cells and fibers of the entopeduncular nucleus are seen in the lateral zone of ventral telencephalic

area (Vl). D: Dense immunoreactivity is seen in the preoptic area (POA) inclusive of magnocellular preoptic nucleus (PM), posterior part of

parvocellular preoptic nucleus (PPp), and some in the suprachiasmatic nucleus (SCN). E: Moderate immunoreactivity is seen in the poste-

rior commissure (PC) and the anterior thalamic nucleus (A). F: Scattered fibers can be seen in the periventricular nucleus of posterior

tuberculum (TPp ) and the ventral zone of periventricular hypothalamus (Hv). G–I,K: Intensely stained CART-positive cells and fibers are

seen in the central posterior thalamic nucleus (CP; G), nucleus of the medial longitudinal fasiculus (N; H), periventricular gray zone of optic

tectum (PGZ; I) and the caudal zone of periventricular hypothalamus (Hc; K). J: A dense network of fibers is seen in the saccus vasculosus

(SV). L: The cerebellum shows scattered fibers in the corpus cerebelli (CC) and the medial division of valvula cerebella (Vam). M–O: CART-

positive cells and fibers are seen around the brachium conjunctivum (BC; M), in the superior raphe (SR; N), and in the facial lobe (LVII; O).

Beaded fibers are seen in the superior reticular formation (SRF; N). P: In the spinal cord, scattered fibers can be seen in the dorsal horn

(DH), funiculus lateralis pars dorsalis (Fld), ventral horn (VH), and funiculus ventralis (Fv). For abbreviations, see list. Scale bars ¼ 50 lmin A,B,D–F,J,L,P; 20 lm in C,G–I,K,M–O.



development, a few CART fibers persisted in the AC of

the adult (Figs. 11D, 13E, 14B). In the ventral telenceph-

alon, several fibers were seen in the lateral (Vl), dorsal

(Vd), and ventral zones (Vv) and the supracommissural

nucleus of the ventral telencephalon (Vs) of the 30 dpf

larva and older fish (Figs. 11C,D, 12C, 13C–E, 14C,D).

The adult fish showed a cluster of three to four immuno-

reactive neurons in the dorsal (Vd) and ventral (Vv)

zones of the ventral telencephalon, but these were not

detected at earlier stages (Figs. 13C,D, 14C).

Figure 13. Schematic drawings of CART immunoreactivity in the CNS of zebrafish at 6 months. Drawing at upper left shows lateral view

of the brain indicating the levels (A–R) represented in the corresponding transverse sections showing CART-immunoreactive cells (dark

circles) and fibers (dots). For abbreviations, see list. Scale bar ¼ 1,000 lm in drawing, upper left; 50 lm in P (applies to A–R).

Mukherjee et al.


CART in the diencephalonThe presumptive POA could be delineated at 4 dpf,

and it showed few CART fibers. However, POA was

clearly defined in the 7 dpf larva and showed CART

fibers organized in discrete agglomerations in the

medial POA that appeared to spread laterally (Figs.

6C,D, 7E). A similar profile was witnessed in all stages

until adulthood (Figs. 9D–F, 10C,E, 11D,E, 12D, 13E–

G, 14B,E,F). In the 4 dpf larva, several immunoreactive

fibers were seen in the poc (Fig. 4D, 5E). This fiber

density peaked at 7 dpf and subsequently decreased,

although some fibers persisted until adulthood (Figs.

6D, 7E, 11F, 13G,H). The thalamus of 4 dpf larva

showed fibers in the dorsal and ventral regions and in

the PC (Figs. 4C,D, 5D,E). In the 7 dpf larva, an arc of

intensely stained fibers passed through the PC and

seemed to bilaterally connect the future preglomerular

complex (migrated posterior tubercular area; M1 as

Figure 13. Continued



Figure 14. Rostrocaudal series of transverse sections (A–K) of CNS of zebrafish at 6 months showing CART-immunoreactive cells (arrows)

and fibers (arrowheads). A: CART-positive fiber distribution in the various parts of the dorsal telencephalon: dorsal zone (Dd), central zone

(Dc), and lateral zone (Dl). B: Immunoreactive fibers are seen in the anterior commissure (AC) and preoptic area (POA). C: Scattered cells

and fibers are seen in the dorsal (Vd) and ventral (Vv) zones of the ventral telencephalic area. D: Intensely stained cells and fibers are

seen in the entopeduncular nucleus (EN) of the lateral zone of ventral telencephalon (Vl). E,F: A network of fibers is seen in the preoptic

area (POA) in the vicinity of the posterior part of parvocellular nucleus (PPp) and beaded fibers are seen in the optic chiasma (OC). G,H:

CART-positive fibers are seen in the pineal gland (E; G) and the lateral part of the habenula (Ha; H). I: Immunoreactive cells and fibers are

seen in the ventromedial thalamic nucleus (VM). J: Beaded fibers are seen in the posterior commissure (PC) and the anterior thalamic nu-

cleus (A). K: Intensely immunoreactive cells and fibers are also seen in the central posterior thalamic nucleus (CP; K). For abbreviations,

see list. Scale bar in K ¼ 50 lm in A,B,E–H; 20 lm in C,D,I–K.

Mukherjee et al.


Figure 15. Rostrocaudal series of transverse sections (A–L) of CNS of zebrafish at 6 months showing CART-immunoreactive cells (arrows)

and fibers (arrowheads). A–D: Intensely immunoreactive cells and fibers are seen in the periventricular gray zone of optic tectum (PGZ; A),

nucleus of the medial longitudinal fasiculus (N; B), dorsal tegmental nucleus (DTN; C), and Edinger-Westphal nucleus (EW; D). A,E: Scat-

tered fibers are also seen in the stratum album centrale (SAC; A) and the pituitary gland (Hy; E). F: A few immunoreactive cells and fibers

are seen in the torus semicircularis (TS). G: Dense fiber network is seen in the caudal zone of periventricular hypothalamus (Hc). H: In the

diffuse nucleus of the inferior hypothalamic lobe (DIL) and the saccus vasculosus (SV), scattered immunoreactive fibers are seen. I–K:

Strong immunoreactivity is seen in the cells and fibers of the superior raphe (SR; I), brachium conjunctivum (BC; J), and facial lobe (LVII;

K). L: In the spinal cord, several fibers are seen in the funiculus ventralis (Fv). For abbreviations, see list. Scale bar in L ¼ 50 lm in

E,G,H,L; 20 lm in A–D,F,I–K.



described by Mueller and Wullimann, 2005; Figs. 6D,E,

7F). The preglomerular nucleus (PG) was clearly identi-

fied at 15 dpf. The PC and the PG area continued to

show CART fibers until adulthood, although decreased

in intensity (Figs. 9G, 10F, 11F–H, 12E 13I,J, 14J).

CART neurons were seen in the ventromedial nucleus

(VM), although their appearance was restricted to the

6 m stage (Figs. 13I, 14I).

The sections through the tectal-thalamic area showed

an intensely stained group of immunoreactive neurons at

the 4 dpf stage (Figs. 4E, 5G). This seems to represent

the central posterior thalamic nucleus (CP) noted in the

30 dpf and adult zebrafish (Mueller and Wullimann,

2005). The neurons in this group showed CART in all

stages until adulthood (Figs. 6E, 7G, 9G,H, 10I, 11G, 12G,

13J, 14K). CART fibers were seen in the rostral or interme-

diate zone of the periventricular hypothalamus (Hr/Hi)

and in the lateral hypothalamus (LH) of 4 dpf larva (Fig.

4D,E). In the 7 dpf larva, diffuse nucleus of the inferior

hypothalamic lobe (DIL) could be identified and showed

scattered CART fibers, which could be followed until

adulthood (Figs. 6F, 9J, 11H–K, 13J–O, 15H). At 15 dpf

and all later stages, the caudal zone of the periventricular

hypothalamus (Hc) showed dense CART fibers (Figs. 9I,J,

10G, 11H,I, 12K, 13K–M, 15G).

At all stages from 30 dpf onward, varying degrees of

CART fibers were seen in the diencephalic areas including

the anterior thalamic nucleus (A), periventricular nucleus

of posterior tuberculum (TPp), magnocellular preoptic nu-

cleus (PM), posterior part of the parvocellular preoptic nu-

cleus (PPp), suprachiasmatic nucleus (SCN), and ventral

and caudal zones of the periventricular hypothalamus

(Figs. 11E–I, 12D–F,K, 13F–M, 14E,F,J, 15G). Immunore-

active fibers appeared in the horizontal commissure (HC),

becoming established at 30 dpf, and could be followed

through until adulthood (Figs. 11F, 13H). One or two

CART-positive somata appeared in the tuberal area (Hc)

of the 30 dpf and older zebrafish (Figs. 11I, 12K, 13K).

Although the periventricular hypothalamic region showed

crowding of somata, these areas were strikingly devoid of

CART. The density of fibers in the lateral tuberal areas

increased with age, and numerous CART fibers were seen

in the Hc region of the hypothalamus. CART fibers were

seen in the saccus vasculosus (SV) of the 30 dpf and

adult fish (Figs. 11J, 12J, 13M, 15H). In the pituitary (Hy)

of developing zebrafish, stray CART fibers first appeared

at 15 dpf and were seen until adulthood (Figs. 9J, 10G,

13J–L, 15E).

Whereas isolated CART fibers were seen in the pineal

gland of fish from 4 dpf (Figs. 4C, 5C) until adulthood

Figure 16. A: NPY-positive neurons (arrows) in the endopeduncular nucleus (EN) region. B: CART-positive neurons (arrowheads) in the EN

region. C: Co-localization of CART and NPY immunoreactivity in the EN neurons (thick arrows). D,E: CART immunoreactivity in the EN neu-

rons of control- (D) and glucose-treated (E) zebrafish at 15 dpf. Note the increase in the immunoreactivity in cells (arrows) and fibers

(arrowheads) of glucose-treated fish compared with control treatment. F: The number of CART-positive cells increased significantly (P <

0.0001) following glucose treatment (E). Values are shown, after Abercrombie correction, as mean 6 SEM (n ¼ 6 brains in each group).

*, P < 0.0001 versus control (first bar). Scale bar ¼ 20 lm in A–E.

Mukherjee et al.


(Figs. 11E, 13F, 14G), CART-positive cells were seen in

the 7 and 15 dpf larvae (Figs. 6D, 7D, 9E, 10H). Fibers

were also seen in the habenula (Ha) from 7 dpf onward

(Figs. 9E,F, 11E, 13G,H 14H).

CART in the midbrainIn the TeO of 4 dpf fish, isolated immunoreactive fibers

were seen in the presumptive outer (stratum marginale

[SM] and stratum opticum [SO]) and inner layers (stratum

album centrale [SAC]; Figs. 4E, 5E). The fibers became

numerous as development progressed, and in the 30 dpf

fish, they were distinctly segregated in the SM/SO, stra-

tum fibrosum et griseum superficiale (SFGS), and SAC

(Figs. 6E,F, 8A, 11G–J, 13I–O, 15A). CART neurons were

detected in the periventricular gray zone of the optic tec-

tum (PGZ) of the 30 dpf juvenile fish, and in all the subse-

quent stages. Some cells of the PGZ showed fibers radi-

ally directed toward the outer layers of the TeO. In the 4

dpf larva, in the area of transition between the midbrain

and hindbrain, intensely stained CART neurons could be

detected along the ependymal lining and seemed to rep-

resent N (Figs. 4E, 5H). These cells, generally three to

four per section, continued until adulthood (Figs. 6F, 8B,

9I, 10J, 11H, 12H, 13K, 15B). Stray fibers were seen in

the torus semicircularis (TS) and torus longitudinalis (TL)

from 7 dpf onward (Figs. 6F,G, 11G–K, 13J–O, 15F). CART

cells in the TS were observed only at the adult stage

(Figs. 13K,M, 15F). Several cells in the dorsal tegmental

nucleus (DTN) showed immunoreactivity only at the 6 m

stage. These might represent the DTN (Figs. 13J,K, 15C)

and the Edinger-Westphal nucleus (EW; Figs. 13J, 15D),

although in the caudal sections they were restricted to

the DTN. Although a few fibers were seen in the pretectal

area of the fish until 30 dpf, the later stages showed con-

sistent increase in fiber density (Figs. 11H,I, 13K–M).

CART in the hindbrain and spinal cordAlthough the cerebellar plate is identifiable at 48 hpf

and is clearly defined by 4 dpf, stray CART fibers are

detected in this region from 7 dpf onward (Figs. 6H, 9J,K,

11H–L, 12L, 13K–P). Several CART-positive cells were

seen in the facial lobe (LVII) of 30 dpf fish and in later

stages (Figs. 11M, 12O, 13Q, 15K).

Two or three neurons were detected in the superior

raphe nucleus (SR) of the 15 dpf larva and in all subse-

quent stages (Figs. 9–11K, 12N, 13O, 15I). As noted at

48 hpf, isolated cells were seen in the caudal portion of

the MO of the 4, 7, and 15 dpf fish (Figs. 4F, 5I,J, 6H, 8D,

9K). At 30 dpf and adult stages, some CART-positive cells

were seen in the vicinity of the brachium conjunctivum

(BC) of the MO (Figs. 11L, 12M, 13P, 15J). A dense net-

work of fibers was seen in the dorso- and ventrolateral

areas of the medulla from 4 dpf onward (Figs. 4F, 5I,J,

6H,I, 8D, 9J,K, 11M, 13P,Q). In the SC, although CART

perikarya were not seen, fibers were detected from 48

hpf onward. At the earlier stages they were confined to

the lateral SC, but in the later stages, fine, often varicose,

fibers were seen throughout (Figs. 4G, 5J, 6J, 8E, 9L, 10L,

11N, 12P, 13R, 15L).

CART in the retinaAlthough no immunoreactivity was seen in the retina of

the 24- or 48-hpf larvae, fish from 4 dpf onward until

adulthood showed CART immunoreactivity in the ama-

crine cells; some fibers were also seen in the inner plexi-

form layer (Figs. 4C,D, 5F, 6K, 7F, 9M, 10B,C). Parallel

varicose fibers were organized in the ganglion cell layer of

all stages from 4 dpf onward (Figs. 4B–D, 5F, 6K, 7F, 9M,

10B,C). Stray CART fibers were seen in the optic nerve

(ON) from 7 dpf onward and in the optic chiasma (OC)

from 15 dpf onward, and these persisted until adulthood

(Figs. 9F, 10C, 11E, 13F, 14F).

Response of the CART neurons in the EN toglucose treatment in developing zebrafish

CART-positive neurons were prominently detected in

the presumptive EN of the developing zebrafish from 7

dpf onward, and by 15 dpf, the EN appeared as a well-

defined cluster. Because EN neurons in most teleosts

contain NPY (Mathieu et al., 2002; Gaikwad et al., 2004;

MacDonald and Volkoff, 2009), we performed dual immu-

nofluorescence analysis with NPY and CART antibodies to

confirm the identity of these cells. The EN at 15 dpf con-

tained several NPY-positive neurons, and CART was found

to be co-expressed in some of these (Fig. 16A–C).

The role of CART in energy metabolism is well known in

mammals as well as in teleosts (Lambert et al., 1998;

Subhedar et al., 2011). With a view to find out if the

CART-containing system participates in the regulation of

energy metabolism early in development, we subjected

the 15 dpf zebrafish to glucose-enriched medium. Rela-

tive to control treatment, glucose immersion caused an

increase in the CART-positive fibers and perikarya of the

EN (Fig. 16D,E). After Abercrombie correction, the aver-

age number of CART-positive neurons in the EN of control

fish was 2.21. Following glucose treatment, this

increased approximately three-fold (6.10, P < 0.0001;

Fig. 16F).

DISCUSSION

To our knowledge, this is the first exhaustive study on

the development of CART in the CNS of a vertebrate spe-

cies. The CART systems in different parts of the brain

seem to become established as early as 24 hpf and

show increasing complexity as development proceeds.



Although the early appearance of the peptide indicates

that it might have a role in the development of brain per

se, the extensive distribution of CART in the zebrafish

brain at 30 dpf and beyond suggests that the peptide

might be mediating activities ranging from processing

sensory information to motor control. The development of

the CART-immunoreactive system, in each component of

brain, is discussed below.

CART in the olfactory systemCART immunoreactivity was detected in the fibers of

the presumptive OB as early as 48 hpf. Olfactory glomer-

uli are established by 4 dpf and show CART immunoreac-

tivity from this stage and continue until adulthood. The

data suggest that CART may play a role in modulating the

olfactory information as it is conveyed from the olfactory

receptor neurons to the mitral cells. It appears that the

CART elements, putatively involved in olfactory process-

ing, are established well before free feeding commences.

CART immunoreactivity was also detected in the olfactory

glomeruli of the adult catfish (Singru et al., 2007). How-

ever, unlike in catfish (Singru et al., 2007) and frog (Lazar

et al., 2004), in which neurons in the granule cell layer in

the OB showed CART immunoreactivity, none were seen

in zebrafish. No CART-containing neurons were seen in

the olfactory organ. Although dense fascicles of CART

fibers were seen in the AC at 48 dpf through 15 dpf, con-

siderable reduction was noted in further stages. Because

the AC is known to bear fibers from the olfactory bulbs

with reciprocal projections (Miyasaka et al., 2009), we

speculate that the rich occurrence of CART fibers, during

early stages, may reflect their involvement in establishing

this commissural pathway.

There appears to be a common theme in the role of

CART in processing olfactory information in vertebrates.

In teleosts, the involvement seems to be at the level of

glomeruli (present study; Singru et al., 2007), whereas in

frog CART seems to influence the inputs at the level of

the mitral cells (Lazar et al., 2004). However, in rat, CART

is observed in mitral cells (Koylu et al., 1998) and may be

involved in relaying information from the mitral cells to

the higher order olfactory centers.

CART in the telencephalonAlthough the CART fibers are widely distributed in the

telencephalon, often in discrete locations, CART-immuno-

reactive neurons were observed in a few groups. Conspic-

uous immunoreactivity was seen in the EN, located in the

ventrolateral telencephalon. These neurons first appear

at 7 dpf, and are established as a prominent cluster by 15

dpf. The fibers are particularly conspicuous in the Dc, Dd,

Vl, and Vs, whereas moderate amounts are seen in the

Vv, Vd, Dm, and Dl, and none in Dp. Corresponding areas

in the brain of adult catfish also showed rich innervations

by CART fibers (Singru et al., 2007). Although the origin

of the CART fibers in the telencephalon is unknown, it is

possible that some of these may originate from the EN,

as suggested by Barsagade et al. (2010) in catfish. The

EN neurons showed reciprocal connections with the Dm

in goldfish (Echteler and Saidel, 1981). The neurons of

the EN attract attention also because, in several teleosts,

they show the presence of NPY (Vallarino et al., 1988;

Mathieu et al., 2002) and in the catfish the neurons

co-contain CART and NPY (Singru et al., 2008). Our data

support these observations.

In the zebrafish, both the Vs and Dm show prominent

CART fibers. Although the earlier literature considered

the Vs of teleosts as the homolog of the amygdala of tet-

rapods (Demski, 1984; Northcutt, 1995), recent lesion

studies suggest Dm to be the functional equivalent of the

amygdala (Portavella et al., 2004). In frog and rat, CART

fibers, as well as cells, are seen in various subdivisions of

the amygdala. Although the Vv of adult zebrafish showed

few CART-positive neurons, no comparable cells were

noticed in the catfish brain.

CART in the diencephalonThe POA is clearly defined by 7 dpf, and through all the

subsequent stages of development, it shows dense net-

work of CART fibers. The importance of the POA in the

regulation of reproduction in teleosts is well recognized

(Yu et al., 1991; Kah et al., 1993). CART elements in this

area are shown to vary with the status of reproductive

maturity in catfish (Barsagade et al., 2010), and it is pos-

sible that a similar function may be attributed to the

CART fibers in the zebrafish. However, unlike in the cat-

fish, no CART perikarya were seen in the POA of

zebrafish.

CART neurons were encountered in the VM of zebra-

fish, as also observed in the catfish (Singru et al., 2007)

and frog (Lazar et al., 2004). Incidentally, in other tele-

osts, VM neurons have been shown to express a number

of neuroactive substances like FMRFamide, NPY, and so-

matostatin (Meek and Nieuwenhuys, 1998). The CP of

zebrafish shows CART-immunostained neurons, reminis-

cent of CART expression in analogous areas in the catfish

and frog (Singru et al., 2007; Lazar et al., 2004). Interest-

ingly, the PG, which in our study shows CART fibers, is

known to project to the VM and CP as well as telencepha-

lon in other teleosts (Murakami et al., 1986a,b; Striedter,

1991, 1992; Wullimann and Meyer, 1993).

In addition to the occurrence of CART cells in the pin-

eal of zebrafish at 7 and 15 dpf, fibers were seen at all

subsequent stages of development. To our knowledge,

this is the first report on the presence of CART in the pin-

eal of a vertebrate species. CART has been reported in

Mukherjee et al.


the Ha of catfish (Singru et al., 2007), as also observed in

this region of the zebrafish from 48 hpf onward. In rat, the

lateral Ha also showed the presence of cells and fibers

(Koylu et al., 1998). The role of the habenular ganglion-

pineal complex in photoreception and circadian rhythms

in teleosts is well established (Ekstrom, 1987). The possi-

bility that CART elements play a role in these functions

deserves to be evaluated.

CART fibers were detected in the hypothalamus from 4

dpf onward, and in the Hy from 15 dpf onward. The CART

fiber density in these areas continued to increase as the

fish attained adulthood. Isolated CART-positive cells were

noted in the tuberal area in the 30 day-old juvenile and older

fish. CART elements were also observed in the hypothala-

mus and Hy of the adult catfish (Singru et al., 2007) and

frog (Lazar et al., 2004). In the catfish, CART immu-

noreactivity in the hypothalamus was correlated with

reproductive maturity status (Barsagade et al., 2010).

CART cells have been detected in the anterior Hy of

rat (Koylu et al., 1997), and its role in the regulation

of Hy function has been extensively documented

(Baranowska et al., 2006; Fekete and Lechan, 2006;

Chmielowska et al., 2011). Similarly, CART is promi-

nently found in several hypothalamic nuclei and is

implicated in energy metabolism in the rat (Koylu

et al., 1997; Kristensen et al., 1998; Lambert et al.,

1998). These data, taken together, suggest that CART

may serve as an important neurotransmitter in the

hypothalamus and pituitary of vertebrates.

The CART immunoreactivity in the PC showed an inter-

esting spatiotemporal pattern. The fibers appear in the

PC at 4 dpf and assume considerable prominence at 7

dpf, when they seem to establish reciprocal connectivity

between the regions described as the future PG by Muel-

ler and Wullimann (2005). However, the immunoreactivity

in the fibers of the PC is decreased in the 15 dpf larva,

and a much more limited signal was detected in the PC of

older fish. We may recall that the nucleus preglomerulo-

sus medialis of C. batrachus showed CART cells (Singru

et al., 2007). In view of the observation that the preglo-

merular complex is involved in the processing of acous-

tico-mechanosensory lateral line inputs and tertiary-gus-

tatory information in a range of teleosts (Murakami et al.,

1986a,b; Wullimann, 1988; Striedter, 1991, 1992; Wulli-

mann and Meyer, 1993), a similar role in the zebrafish

may be speculated.

CART in the optic tectum, pretectum, andsynencephalon

CART was consistently seen in the TeO. Initially, fibers

were identified at 4 dpf with the cells appearing at 30

dpf. As reported in the adult catfish and frog, these were

organized in different laminae. A similar pattern has been

reported in the adult catfish and frog (Singru et al., 2007;

Lazar et al., 2004). The TeO of teleosts is known to be the

primary target of the optic nerve fibers and also serve an

important role as a sensory-motor center (Meek and

Nieuwenhuys, 1998). Small CART-positive cells were

seen the TS of the adult zebrafish, catfish, and frog (Lazar

et al., 2004; Singru et al., 2007). The TS is suggested to

be involved in acoustic, visual, and mechanosensory lat-

eral line inputs in teleosts (Page and Sutterlin, 1970; Ech-

teler, 1985; Nederstigt and Schellart, 1986). The pres-

ence of CART neurons and a rich fiber population in this

region, taken together with the CART-positive elements in

the PG, suggests a role for the neuropeptide in these

functions. CART-immunoreactive neurons were seen in

the DTN and EW of the zebrafish, and anatomically these

seem similar to the dorsal posterior tegmental nucleus

described by Singru et al. (2007). CART is known to be

present in the EW of vertebrate species belonging to dif-

ferent phylogenetic groups (Koylu et al., 1998; Lazar et

at., 2004). In teleosts, the EW is involved in accommoda-

tion and pupillary light reflex (Nilsson, 1980; Wathey,

1988). It is therefore possible that CART in the EW of

adult zebrafish may play a similar role.

The 24 hpf larva showed a small group of CART-

positive neurons in the sections as well as whole

mounts, in an area corresponding to the vcc, which is

known to be the precursor of the N (Geling et al.,

2003). In the zone of transition between the midbrain

and hindbrain, CART-positive neurons were observed in

the N at 4 dpf; the cells were more numerous in the

15 dpf zebrafish, and continued to be detected in the

adult. The N issues processes that form the MLF, and

a CART-ergic contribution to this tract was observed

as early as 24 hpf. In view of their location on the

periventricular face, these neurons are comparable to

the CART-containing neurons in the synencephalon of

adult catfish (Singru et al., 2007). CART cells were

also reported in the N of frog (Lazar et al., 2004).

CART in the medulla oblongata, cerebellum,and spinal cord

As reported in the adult catfish, frog, and rat (Koylu

et al., 1998; Lazar et al., 2004; Singru et al., 2007), CART

neurons are seen in the SR of zebrafish from 15 dpf

onward. Although CART-positive cells in the BC appear at

30 dpf, it is possible that they are derived from the CART

neurons that appeared in the dorsal MO at earlier stages.

Few CART neurons were seen in the LVII, and these seem

analogous to the facial motor nucleus (FMN) described in

the catfish and frog (Lazar et al., 2004; Singru et al.,

2007).



Although the cerebellar plate is identifiable in the 48

hpf larva, CART fibers were detectable in the cerebellum

from 7 dpf onward. These fibers localize in the granule

cell layer similar to the pattern reported in the frog (Lazar

et al., 2004). Interestingly, in the adult catfish, fibers as

well as cells were observed in the granule cell layer

(Singru et al., 2007). In rodents, CART immunoreactivity

has been reported in the climbing and mossy fibers of the

cerebellum (Press and Wall, 2008; Reeber and Sillitoe,

2011). In the SC of zebrafish, whereas CART fibers are

detected from 48 hpf onward, no cells were observed at

any stage. In the frog, CART cells were reported in the

dorsal horn and fibers elsewhere (Lazar et al., 2004),

whereas in the rat, cells were seen in the thoracic SC and

fibers in the dorsolateral fasciculus (Koylu et al., 1998).

Role of CART in energy metabolismExposure of the 15 dpf larvae to glucose-enriched me-

dium increased the population of CART cells in the EN by

almost threefold. This suggests that the EN has a popula-

tion of CART-competent neurons that express CART de

novo following glucose treatment. We may recall that in

the adult catfish, exposure to glucose increased the pop-

ulation of CART neurons in the EN, whereas administra-

tion of 2-DG resulted in the loss of CART peptide from the

neurons and the CART-positive fiber system in the telen-

cephalon (Subhedar et al., 2011). These changes reflect

the involvement of the endogenous CART system in proc-

essing energy status-related information. Our data sug-

gest that a similar situation might also prevail in the

zebrafish. Our study indicates that the CART-containing

neurons of the EN may be a part of the circuitry that proc-

esses energy-status related information, as early as 15

dpf. This is not surprising because the zebrafish starts

feeding from about 7 dpf and may depend on this cir-

cuitry to regulate its food intake-related behavior. These

conclusions are consistent with the established view that

CART serves as an anorexic agent in teleosts (Volkoff and

Peter, 2000, 2004; Kobayashi et al., 2008) and also in

mammals (Lambert et al., 1998; Thim et al., 1998; Kuhar

and Dall Vechia, 1999; Larsen et al., 2000).

General considerationsAn overview of the development of CART in the CNS of

zebrafish suggests two trends. First, the CART-immunore-

active elements are overrepresented in the commissural

tracts like the AC, poc, and PC. Although conspicuous

CART fiber aggregation is observed in the early stages

(4–7 dpf), this tends to decrease considerably in all three

sites at the later stages. We speculate that CART may

have a role in the initial establishment of these axonal

tracts. Second, certain CART elements appear late in de-

velopment, suggesting involvement in functions that arise

later. CART-containing perikarya appear in the PGZ, BC,

and LVII on a delayed time scale (30 dpf) and become

established as the fish attains adulthood. In other areas

like the Vv, Vs, VM, DTN, and EW, cells appear only in the

adult. Because zebrafish is known to attain full sexual ma-

turity at about 6 months of age, it is possible that some of

these structures may be involved in puberty or some

other functions that co-occur with sexual maturity.

The occurrence of CART in the olfactory system, ama-

crine cells of the retina, ON, optic lobes, E, Ha, PG, and

TS underscores the possibility that the peptide may be

involved in processing of sensory information. Broadly,

analogous areas in the brain of other vertebrates have

also been shown to contain CART (Koylu et al., 1998;

Lazar et al., 2004; Singru et al., 2007). Several compo-

nents of the visual system inclusive of the retina, optic

nerve, and optic lobes show the presence of CART in dis-

crete elements, right from early stages of development.

The pineal-habenular complex also showed the presence

of CART, as does the EW, which plays a role in accommo-

dation and pupillary light reflexes in teleosts (Nilsson,

1980; Wathey, 1988). Thus a role for the peptide in photic

information processing may be suggested, and future

studies aimed at investigating this area may be

rewarding.

Among teleost fish, systematic study of CART distribu-

tion has been reported in adult catfish (Singru et al.,

2007; Subhedar et al., 2011). Comparison of the CART

distribution between the zebrafish and catfish shows sev-

eral striking similarities, particularly with reference to the

olfactory glomeruli, EN, several areas of the telencepha-

lon, POA, Ha, DT, hypothalamus, Hy, TeO, and hindbrain.

This suggests that the peptide may have similar func-

tional attributes. However, some differences between the

CART profile in catfish and zebrafish were also noted. In

the catfish, CART was enriched in the gustatory system

(Singru et al., 2007), although a similar emphasis was not

observed in zebrafish. It is possible that the gustatory

pathways in the zebrafish may employ other neurotrans-

mitter systems.

The CART distribution pattern across different verte-

brate lineages displays several common features (present

study; Koylu et al., 1998; Lazar et al., 2004; Singru et al.,

2007) and is suggestive of evolutionarily conserved func-

tional trends. The present study suggests that the CART

peptidergic system is not only an important component

of the adult vertebrate CNS, but may also have a critical

role in development. Zebrafish is a genetically amenable

vertebrate model with well-defined developmental trajec-

tories and behavioral paradigms. This work provides the

neuroanatomical underpinnings for future interventional

studies exploring CART function in development and

behavior in this vertebrate.

Mukherjee et al.


ACKNOWLEDGMENTS

The authors are grateful to Lars Thim and Jes Clausen,

Novo Nordisk, Denmark for providing monoclonal anti-

bodies against CART and CART peptide (54–102).

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Ontogeny of the cocaine- and amphetamine-regulated transcript (CART) neuropeptide system in the brain of zebrafish, Danio rerio