Early developmental expression of leptin receptor gene and [125I]leptin binding in the rat forebrain.
ABSTRACT Leptin, via leptin receptors (Ob-R), regulates appetite and energy balance. Of the six isoforms of the receptor identified, so far, only the long form (Ob-Rb) can fully activate downstream signal transduction pathways. Although the expression and function of leptin receptors is well described in the adult brain, little is known about the ontogeny of leptin receptor system around the time of birth. In this study, the mRNA expression patterns of total leptin receptor, Ob-R, and the long signalling form of the receptor, Ob-Rb, were investigated in the brain of embryonic and newborn rats using in situ hybridisation and [125I]leptin binding. On embryonic day 18 (E18), Ob-R mRNA was detected in the choroid plexus and the ependymal layer of the third ventricle by in situ hybridisation. At E21, Ob-Rb mRNA was first observed in the arcuate and the ventral premammillary hypothalamic nuclei while at P3, receptor expression was also found in the dorsomedial nucleus. Other leptin target areas identified were the trigeminal ganglion, the thalamus and the hippocampus. Using quantitative receptor autoradiography specific [125I]leptin binding sites on the choroid plexus were found to increase with age in contrast to the ependymal layer of the third ventricle where levels decreased with age. Together these findings demonstrate that the leptin receptor system is differentially regulated during late gestation and early postnatal life in the rat.
- SourceAvailable from: Pekka MäntyselkäPreventive Medicine - PREV MED. 01/2011; 53(3):211-211.
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ABSTRACT: Brain development is a complex and dynamic process, and many environmental factors have been found to influence the normal development of neural pathways. Cumulative evidence suggests that metabolic hormones that regulate the hypothalamic circuits that control energy homeostasis function in much the same way that sex steroids act on sexually dimorphic circuits. For example, although the effects of the adipocyte-derived hormone leptin were originally thought to be limited to the neural control of energy homeostasis in adult animals, it is now becoming increasingly clear that leptin can also determine patterns of neurogenesis, axon growth, and synaptic plasticity in the developing hypothalamus. More recent studies have also extended the role of the metabolic hormones ghrelin and insulin in various aspects of brain development. Examining how metabolic hormones control hypothalamic development will help our understanding of the developmental origin of adult metabolic diseases and, hopefully, improve our ability to predict adverse outcomes.Frontiers in Neuroendocrinology 01/2013; · 7.99 Impact Factor
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ABSTRACT: Circulating leptin crosses blood-brain barrier to provide control of feeding behavior and energy balance. We investigated changes in leptin and leptin receptor (ObR) in the gerbil hippocampal CA1 region (CA1) after transient cerebral ischemia, and examined effects of leptin on ischemic damage. In vehicle-treated ischemia (vehicle-ischemia) group, the number of survived neurons in the CA1 was 16.4% compared to vehicle-treated sham (vehicle-sham) group; however, in 1 mg/kg leptin-treated ischemia (leptin-ischemia) group, 77.5% of neurons of the CA1 has survived. In the vehicle-sham group, weak leptin immunoreactivity was detected in CA1 neurons. From 4 days post-ischemia, moderate leptin immunoreactivity was expressed in CA1 neurons. In the leptin-ischemia group, leptin immunoreactivity at 5 days post-ischemia was higher than the sham group. ObR immunoreaction in the sham group was hardly detected in any cells. From 2 days post-ischemia, ObR immunoreaction was expressed in microglia, showing the highest immunoreactivity at 5 days post-ischemia. Microglial activation in the leptin-ischemia group was hardly detected at 5 days post-ischemia; however, astrocytes in the group were slightly increased compared to the vehicle-ischemia group. These suggest that treatment of leptin has neuroprotective effects against ischemic damage, showing that ObR immunoreactivity is distinctly changed in the ischemic CA1.Journal of the neurological sciences 01/2011; 303(1-2):100-8. · 2.32 Impact Factor
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HPtg, hamulus of the pterygoid bone; Lep, leptomeninges; LV, lateral ventricle;
T, thalamus; ME, median eminence; PMV, premamillary nucleus, ventral part;
PSph, presphenoid bone; Ptg, pterygoid bone; PH, posterior hypothalamic area;
VMH, ventromedial hypothalamic nucleus
* Corresponding author. Tel.: +44 1224 716 682; fax: +44 1224 716 686.
E-mail address: L.Williams@rowett.ac.uk (L.M. Williams).
Early developmental expression of leptin receptor gene and
[125I]leptin binding in the rat forebrain
Anne-Sophie Carloa,b, Wolfgang Meyerhofb, Lynda M. Williamsa,*
aMetabolic Health Group, Rowett Research Institute, Greenburn Rd, Bucksburn, Aberdeen, Scotland, UK
bDepartment of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany
Received 11 July 2006; received in revised form 22 February 2007; accepted 22 February 2007
Available online 27 February 2007
Leptin, via leptin receptors (Ob-R), regulates appetite and energy balance. Of the six isoforms of the receptor identified, so far, only the long
form (Ob-Rb) can fully activate downstream signal transduction pathways. Although the expression and function of leptin receptors is well
described in the adult brain, little is known about the ontogeny of leptin receptor system around the time of birth. In this study, the mRNA
expression patterns of total leptin receptor, Ob-R, and the long signalling form of the receptor, Ob-Rb, were investigated in the brain of embryonic
and newborn rats using in situ hybridisation and [125I]leptin binding. On embryonic day 18 (E18), Ob-R mRNAwas detected in the choroid plexus
and the ependymal layer of the third ventricle by in situ hybridisation. At E21, Ob-Rb mRNA was first observed in the arcuate and the ventral
were the trigeminal ganglion, the thalamus and the hippocampus. Using quantitative receptor autoradiography specific [125I]leptin binding sites on
the choroid plexus were found to increase with age in contrast to the ependymal layer of the third ventricle where levels decreased with age.
Togetherthesefindingsdemonstratethattheleptin receptorsystemisdifferentially regulatedduringlategestationandearlypostnatal lifeinthe rat.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Leptin; Development; Hypothalamus; In situ hybridisation; Receptor autoradiography
Leptin is the product of the ob gene and is secreted by white
adipose tissue. It regulates energy intake and expenditure by
acting mainly on the hypothalamus, particularly the arcuate
nucleus (ARC) thereby promoting satiety and increasing
energy expenditure (Elmquist et al., 1998b; Schwartz et al.,
2000). Apart from its role in food-intake regulation, leptin is
also involved in the regulation of reproduction, the immune
system (Hoggard et al., 1998; Waelput et al., 2006) and the
neuroendocrine adaptation to food restriction (Ahima et al.,
1996; Casanueva and Dieguez, 1999).
Leptin also appears to act as an important neurotrophic
factor in the brain with mice that lack either leptin (ob/ob) or
functional leptin receptors (db/db) having reduced brain
weights and morphological defects (Bereiter and Jeanrenaud,
1979, 1980; Sena etal., 1985; Garris, 1989;Steppanand Swick,
1999). These observation were confirmed when leptin was
shown to promote the development of projections from the
ARC to other hypothalamic nuclei, implicated in food-intake
regulation, with these projections being severely reduced in the
absence of leptin in ob/ob mice (Bouret et al., 2004a, 2004b).
The actions of leptin are mediated via its receptor, Ob-R, a
class I cytokine receptor, having a single membrane-spanning
domain (Tartaglia et al., 1995), with different isoforms of the
receptor (Ob-Ra-f) being derived by alternative splicing of the
gene and post-translational processing (Chua et al., 1997; Ge
et al., 2002). All isoforms have a similar extracellular ligand-
binding domain at the amino terminus, but differ at the
intracellular carboxy-terminal domain. Only the long form of
the receptor, Ob-Rb, and possibly one shorter isoform, Ob-Ra,
appear capable of signalling and are thus able to mediate the
biological effects of leptin. Ob-Rb has the full signalling
Journal of Chemical Neuroanatomy 33 (2007) 155–163
Abbreviations: 3V, third ventricle; 5Gn, trigeminal ganglion; ARC, arcuate
nucleus; CP, choroid plexus; D3V, dorsal third ventricle; Hi, hippocampus;
0891-0618/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
Author's personal copy
collected onto a set of six slides with adjacent sections on consecutively
numbered slides. Adult brains were sectioned onto a series of ten slides.
Sections spanned the full extent of the ARC, according to the atlas of the
rat brain (Paxinos et al., 1994). For [125I]leptin binding, serial sections (slides 1
and 2 for the embryonic and neonatal brain) were thaw mounted onto 0.5%
poly-L-lysine coated slides. Slides 3–6 of the embryonic and neonatal brain
were used for Ob-Rb and Ob-R in situ hybridisation and their controls,
capacity as it possesses the consensus sequences necessary for
signal transduction (Baumann et al., 1996).
In the adult animal, Ob-R and Ob-Rb gene expression is
detected in several brain areas with high levels of expression in
the hypothalamus (Mercer et al., 1996; Buyse et al., 2001)
localised to the ARC, ventromedial (VMH), dorsomedial
(DMH) and ventral premammillary nuclei (PMV) with lower
levels of expression in the paraventricular nucleus (PVN) and
the lateral hypothalamic area (LHA) (Mercer et al., 1996; Fei
et al., 1997; Elmquist et al., 1998a). Among the short forms of
the leptin receptor, Ob-Ra is the most abundantly expressed,
particularly in the choroid plexus and brain microvessels
(Tartaglia et al., 1995; Mercer et al., 1996; Bjorbaek et al.,
1998) where, along with Ob-Rc, it was thought to function as a
Studies have examined the development of Ob-Rb expres-
sion in the rat and mouse embryonic brain (Matsuda et al.,
1999; Udagawa et al., 2000). However, detailed knowledge is
the newly identified role of leptin in development of the late
embryonic and early postnatal brain. Thus, in the present study,
the expression of Ob-R and Ob-Rb is described using both in
situ hybridisation and [125I]leptin binding in the rat brain in late
gestation and early neonatal life at three developmental stages:
embryonic day 18 (E18), E21 and postnatal day 3 (P3).
2. Materials and methods
Timed-pregnant Sprague-Dawley (CD) rats were purchased from Charles
Institute of Human Nutrition. Animals were housed in individual cages under
standardconditionswitha 12/12 hlight/darkcycle(lightsonat 6a.m.)at22 8C.
Animals had ad libitum access to water and standard lab chow (Altromin,
Altromin GmbH). Timed-pregnant rats on day 18 of gestation (E18; the day of
mating is counted as day 0) and on day 21 were anaesthetised using ketamine
(100 mg/kg body weight) (Albrecht, Aulendorf, Germany) and xylazine
(10 mg/kg body weight) (Rompun, Bayer Vital, Leverkusen, Germany) and
the embryos killed by decapitation. For postnatal rats, the day of birth was
considered as postnatal day 0. Pups were kept undisturbed with their mother
until they were killed by cervical dislocation. Eight animals at the develop-
mental stage E18, six at E21 and seven at P3 were used for the present study.
Animal experimentation complied with the ethical guidelines for the care
and use of laboratory animals of the Ministry of Agriculture for Nutrition and
Forestry (State Brandenburg, Germany, approval number 32/48-3560-0/3).
2.2. Tissue preparation
Whole embryonic and neonatal rat heads and adult rat brains were rapidly
frozen in isopentane chilled over dry-ice and stored at ?80 8C. 20 mm-thick
transverse sections were cut on a Leica CM1900 cryostat (Leica Microsystems
AG, Wetzlar, Germany). For the embryonic and neonatal brain, sections were
2.3. Generation of leptin receptor riboprobes
Receptor-specific PCR primers were used to generate riboprobes for the
common extracellular domain of the leptin receptor sequence, Ob-R, which
recognizes all of the known splice variants and for the long isoform of the
receptor, Ob-Rb,as alreadydescribed (Merceret al., 1998; Murrayet al., 2000).
The Ob-R probe, a 474 bp product, was generated using rat-specific primers 50-
CAGATTCGATATGGCTTAAATGG-30(1705-1727) and 50-GTTAAAATT-
CACAAGGGAGGCA-30(2157-2178; Genbank D84551). Plasmids were line-
arised with SacI or ApaI for transcription with T7 or SP6 RNA polymerase to
generate sense and antisense riboprobes, respectively. The probe specific to rat
Ob-Rb, a 474 bp product, was generated using the primers 50-AGTGAC-
CAGTGTAACAGTGC-30(2863-2882) and 50-YCTGATGTCACTGAACA-
GAC-30(3317-3336; Genbank D84551). Plasmids were linearised with
BamHI or EcoRV for transcription with T7 or SP6 RNA polymerase to generate
sense and antisense riboprobes, respectively. [35S]-UTP sense and antisense
riboprobes were prepared from 1 mg linearised plasmid DNA.
2.4. In situ hybridisation
In situ hybridisation with Ob-R and Ob-Rb specific riboprobes was
sectionswere fixedin4%PFAin0.1 MPBfor 20 minat 4 8C andthenwashed
twicein0.1 MPB.Sectionswereincubatedin0.1 mmol/Ltriethanolaminefor
2 min and acetylated in 0.1 mmol/L triethanolamine and 0.25% (v/v) acetic
anhydride for 10 min at room temperature followed by washing in 0.1 M PB.
Sections were then dehydrated through a graded series of ethanol, vacuum-
dried and then incubated with riboprobes at 104cpm/mL for 18 h at 58 8C.
Sections were then washed in 4X SSC for 30 min at room temperature. Non-
hybridised RNA was digested with 20 mg/mL ribonuclease A at 37 8C for
stringency of 0.1X SSC at 60 8C for 30 min and finally dehydrated in ethanol.
Air-dried slides were apposed to Hyperfilm bmax (Amersham Biosciences,
(Amersham Biosciences) for 2 weeks (Ob-R) or 6 weeks (Ob-Rb) at room
temperature. Sections were counterstained with thionine to delineate the
margins of nuclear boundaries. Sections are correlated to the nearest appro-
priate section in the Atlas of the Developing Rat Nervous System (Paxinos
et al., 1994) and also to the nearest appropriate section in the adult rat brain
(Paxinos and Watson, 1986).
2.5. Light microscopy
Slides were coated with LM-1 photoemulsion (Amersham Biosciences)
according to manufacturer’s instructions. Briefly, the LM-1 photoemulsion was
incubated at 42 8C for 30 min and apposed to the slides with a loop. Slides were
then air-dried for 30 min and stored at 4 8C prior to development. After 12
for 4 min, followed by 30 s in distilledwater, 4 min in Kodak Unifixand 30 min
in distilled water. Slides were then air-dried, dipped in solvent CNP-30 (Taab
Laboratories, Aldermaston, U.K.) and mounted in DPX (Agar Scientific,
Stansted, U.K.). Silver grains were visualised using a Leica DMR light
microscope (Leica Microsystems AG). Images were captured by a Hamamatsu
software (Media Cybernetics, Wokingham, Berkshire, U.K.).
2.6. [125I]Leptin binding
20 mm-thick coronal hypothalamic cryosections were brought to room
acid-wash buffer at 4 8C for 6 min in order to dissociate bound leptin from the
receptor. Sections were then washed in 100 mM HEPES buffer at pH 7.4 for
2 ? 2 min then incubated with 1 nM [125I]leptin (Perkin Elmer Life Sciences,
Bucks, U.K.) with the specific activity adjusted to approximately
250,000 cpm/pmol in HEPES buffer for 2 h at room temperature. Sections
were then washed 4 ? 5 min in HEPES buffer at 4 8C and briefly rinsed in
distilled water to remove salts before being air-dried and apposed to Kodak X-
A.-S. Carlo et al./Journal of Chemical Neuroanatomy 33 (2007) 155–163156
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riboprobes (not shown).
At E21, [125I]leptin binding appears over the choroid plexus,
leptomeninges, trigeminal ganglion and the ependymal layer of
the third ventricle but binding over hypothalamic neuronal
areas was not observed (Fig. 2F and H). The highest levels of
Ob-R gene expression are also found over the choroid plexus,
leptomeninges, trigeminal ganglion and the ependymal layer of
AR OMAT film (Sigma) together with [125I] microscales (Amersham) for 6
For [125I]leptin binding, mean optical densities of choroid plexus and of the
ependymal layer of the third ventricle were measured using the Image Pro Plus
system (Media Cybernetics, Wokingham, U.K.) and converted to nCi/mg tissue
minimally adjusted in contrast and brightness for illustrative purposes using
Adobe Photoshop 7.1.1. (Adobe Systems, Mountain View, CA, USA).
2.8. Statistical analysis
Results were analysed by one-way ANOVA using Genstat statistical soft-
using Student-Newman-Keuls multiple range test. The level of significancewas
set as P < 0.05. All values are expressed as mean + S.E.M.
In the adult rat brain specific [125I]leptin binding is most
heavily concentrated over the choroid plexus of the lateral and
third dorsal ventricles. Lower levels of binding occur over the
thalamus (Fig. 1A). In agreement with previous published data
(Elmquist et al., 1998a), in situ hybridisation in the adult rat
brain, using a riboprobe specific for Ob-R, shows binding to the
choroid plexus of the lateral and third dorsal ventricles, the
hippocampus, the thalamus and the hypothalamus (Fig. 1B).
Ob-Rb gene expression correlates closely with that of Ob-R
apart from much lower levels of signal detectable over the
choroid plexus (Fig. 1C). No signal is apparent when sections
are hybridised with the equivalent sense probes (not shown).
The lack of signal seen with the sense riboprobes together with
the identical localisation of Ob-R and Ob-Rb riboprobe
labelling (Elmquist et al., 1998a) validate the specificity of
the Ob-R and the Ob-Rb riboprobes used in this study. These
probes have also been used previously to localise leptin
receptor gene expression in the rodent hindbrain (Mercer et al.,
[125I]Leptin autoradiography with E18 brain sections shows
specific binding over the choroid plexus, leptomeninges,
trigeminal ganglion and ependymal layer of the third ventricle
(Fig. 2A and C). No non-specific [125I]leptin binding was
observed in the presence of 10?6M leptin (not shown). In
agreement with the pattern of [125I]leptin binding the highest
levels of Ob-R hybridisation signal are present over the choroid
plexus, leptomeninges, ependymal layer of the third ventricle
and trigeminal ganglion (Fig. 2B and D). Ob-Rb hybridisation
signal over the lateral ventricles is comparatively less intense
than Ob-R expression over this area (Fig. 2E). No hybridisation
signal was detected with either the Ob-R or the Ob-Rb sense
the third ventricle (Fig. 2G and I) as seen at E18. In the
hypothalamus, Ob-R mRNA was evident throughout the ARC
(Fig. 2G and I). The localisation of Ob-Rb mRNA in neuronal
areas was similar to that of Ob-R, in the ARC and in the
posterior hypothalamus (PH) (Fig. 3A). Relativelylowlevelsof
hybridisation signals were evident over the choroid plexus
and the walls of the lateral ventricles (Figs. 2J and 3A). Also,
at E21, Ob-Rb mRNA was detected over the ventral
Fig. 1. Representative sections showing (A) [125I]leptin binding, (B) Ob-R
mRNA expression and (C) Ob-Rb mRNA expression in the adult rat brain.
[125I]Leptin binding is present in the choroid plexus of the lateral ventricle (LV)
and the third dorsal ventricle (D3V) and over the ventroanterior and ventro-
posterior nuclei of the thalamus (T). Ob-R gene expression is observed over the
choroid plexus and the thalamus as detailed above and over the hippocampus
(Hi) and hypothalamic areas including the ventromedial (VMH) and arcuate
(ARC) nuclei. Ob-Rb ISH signals are detected in the hippocampus, thalamus
and hypothalamus as detailed above but are very low or absent over the choroid
plexus. Bar = 2.1 mm.
A.-S. Carlo et al./Journal of Chemical Neuroanatomy 33 (2007) 155–163157
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riboprobes are shown once illustrating a single area of non-specific binding found (3B). Identification of areas is generally limited to the Ob-R figures to provide
clarity and visibility of labelled structures. However, areas are indicated if not present in the Ob-R figures. E18 (A and C) [125I]leptin binding is seen over the
ependymallayerofthe thirdventricle(3V),thetrigeminalganglion(5Gn)andthe choroidplexusofthe lateralventricle (LV) andthe dorsalthirdventricle(D3V) and
the leptomeninges (Lep). (B, D and E) Ob-R gene expression is shown in the trigeminal ganglion (5Gn). Ob-R and Ob-Rb mRNAwas expressed in the ependymal
layer of the third ventricle (3V) and in the walls of the lateral ventricle (LV). Bar = 1.7 mm. Corresponding dark-field image of boxed area in (E) shows Ob-Rb
expression overthe ependymallayerofthe thirdventricle (3V).Bar = 170 mm.E18(A and B)correspondto embryonicE17coronalsection5 andadult brainbregma
?0.92.E18(C,DandE)correspond toembryonicE17coronalsection9andadultbrainbregma?4.30.E21(FandH)[125I]leptinbindingisseenover theependymal
layer of the third ventricle (3V), the trigeminal ganglion (5Gn) and the choroid plexus of the lateral ventricle (LV) and the dorsal third ventricle (D3V) and the
binding did not show any non-specific binding in the presence of 10?6M leptin and are therefore not shown. Similarly in situ hybridisation controls using sense
A.-S. Carlo et al./Journal of Chemical Neuroanatomy 33 (2007) 155–163158
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apparent over the ventral premamillary nuclei (PMV) as well as the ependymal layer of the third ventricle (3V). Bar = 1.8 mm. Corresponding dark-field image of
boxed area in (J) shows Ob-Rb expression over the ependymal layer of the third ventricle (3V). Bar = 180 mm. Hypothalamic areas expressing Ob-Rb at E21 are
embryonicE19coronal section 29 and adult brain bregma ?3.80.P3 (K and N) [125I]leptin binding is seen in the trigeminalganglion(5Gn) and the choroid plexus of
the lateral ventricle (LV) and the dorsal third ventricle (D3V), the developing presphenoid bone (PSph), the pterygoid bone (Ptg) and faintly over the arcuate nuclei
(ARC). (L and O) Ob-R mRNA expression in the choroid plexus of the lateral ventricle (LV) and the dorsal third ventricle (D3V), the hippocampus (Hi) and the
arcuate nuclei (ARC). (M and P) Ob-Rb gene expression is also apparent over the ventral premamillary nuclei (PMV) as well as the ependymal layer of the third
ventricle (3V) the hippocampus (Hi) and the arcuatenuclei (ARC).Bar = 2.0 mm. P3 (K and L) correspond to postnatal 0–16and adult brain bregma ?0.80.E21 (M)
corresponds to postnatal 0–39 and adult brain bregma ?3.80. (N, O and P) corresponds to postnatal 0–36 and adult brain bregma ?3.60.
premammillary nucleus of the hypothalamus (PMV) (Fig. 2J).
Photomicrographs confirmed Ob-Rb expression at the cellular
level in the ependymal layer of the third ventricle, the ARC and
the PH (Figs. 2J, 3A and 4).
At P3, [125I]leptin binding is detected over the choroid
plexus, leptomeninges, trigeminal ganglion and ependymal
layer of the third ventricle (Fig. 2K and N) with comparatively
low levels of [125I]leptin binding now apparent over the ARC
(Fig. 2N). [125I]leptin binding was not observed over other
hypothalamic areas. Specific [125I]leptin binding was also
observed in the presphenoid bone and the pterygoid bone
(Fig. 2N). The pattern of Ob-R and Ob-Rb gene expression was
similar to that seen at earlier developmental stages but with
expression becoming stronger in neuronal areas (Fig. 2L–P).
Ob-R gene expression was again observed in the PMV
(Fig. 2M) and relatively weak Ob-R and Ob-Rb gene
leptomeninges (Lep). E21 (G and I) Ob-R mRNA expression in the ependymal layer of the third ventricle (3V) and in the choroid plexus of the lateral ventricle (LV)
and the dorsal third ventricle (D3V) and the leptomeninges (Lep). Gene expression is also apparent over the arcuate nuclei (ARC) (J) Ob-Rb gene expression is also
hypothalamus (PH)at E21.Seefigure3Bforbar.Correspondingandthioninestainedlight-fieldanddark-fieldimagesofboxedarea. Bar = 160 mm.(B)Non-specific
binding after hybridisation with the Ob-Rb sense probe is shown over the developing presphenoid bone (PSph), the pterygoid bone (Ptg) and the hamulus of the
pterygoid bone (HPtg). Bar = 1.2 mm. Sections correspond to embryonic E19 coronal section 19 and adult brain bregma ?3.60.
Fig. 4. (A) E18 (B) E21 (C) E18. Light-field thionine stained sections demonstrating autoradiography for Ob-Rb mRNA expression over the ependymal layer of the
third ventricle (3V) and the arcuate nucleus (ARC). Median eminence (ME). Bar = (A) 9.6 mm (B) 29 mm (C) 29 mm.
A.-S. Carlo et al./Journal of Chemical Neuroanatomy 33 (2007) 155–163 159
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expression was found over the dorsomedial nucleus (DMH)
The sites of the leptin receptor (Ob-R and Ob-Rb) gene
expression and the [125I]leptin binding at the developmental
stages examined are summarised in Table 1.
Quantitative analysis of [125I]leptin binding, to the choroid
plexus of the lateral ventricle and the dorsal third ventricle
revealed significant changes with developmental age (Fig. 5).
[125I]leptin binding in the choroid plexus of the lateral ventricle
was maximal at P3 (E18 versus P3, P < 0.001; E21 versus P3,
P < 0.001). The same pattern was observed in the dorsal third
ventricle. No significant differences were found between E18
and E21. A different pattern in [125I]leptin binding was
observed over the ependymal layer of the third ventricle with a
decrease of binding detected at P3 (E21 versus P3, P < 0.01).
The present study provides a detailed description of the
development of Ob-R and Ob-Rb in late embryonic and early
postnatal development using in situ hybridisation matched with
[125I]leptin receptor autoradiography. In the adult rat brain,
high levels of Ob-Rb gene expression have been demonstrated
using in situ hybridisation in the ARC, DMH, PVN and VMH
of the hypothalamus, regions that are known to be primary sites
for the regulation of energy balance by leptin (Mercer et al.,
1996; Fei et al., 1997). In previous studies Ob-Rb mRNA was
detected by in situ hybridisation in the ARC and the VMH at
E18.5 in mice (Udagawa et al., 2000) and weak Ob-Rb
immunoreactivity has been reported in the PVN at E18
(Matsuda et al., 1999). In the present study, hypothalamic
nuclei were devoid of Ob-R or Ob-Rb gene expression using in
the ARC and the PMVat E21 and Ob-Rb mRNAwas identified
in the ARC, PH, PMV and DMH at P3. These observations
indicate that there is a sequential appearance of increasing
levels of leptin receptor expression in the developing
hypothalamus starting in the ARC between E18 and E21.
There was no detectable [125I]leptin binding to the
hypothalamic nuclei at most of the developmental stages
tested although a weak [125I]leptin signal was observed in the
ARC at P3, indicating leptin receptor ability to bind ligand. A
similar absence of leptin binding in the hypothalamus has been
described previously in the neonatal and adult brain (Devos
et al., 1996; Lynn et al., 1996; Malik and Young, 1996; Dal
Summary of leptin receptor expression in embryonic and newborn rats
Ob-R mRNA Ob-Rb mRNA[125I]Leptin binding
E18E21P3 E18E21P3 E18E21P3
Ventroanterior and ventroposterior nuclei
Ependymal layer (3V)
Choroid plexus (LV)
Choroid plexus (D3V)
Lateral ventricle walls
At E18, Ob-R and Ob-Rb mRNA is detected in the ependymal layer of the third ventricle whereas at E21, it is expressed in the arcuate nucleus (ARC), the posterior
hypothalamus (PH) and in the ventral premamillary nucleus of the hypothalamus (PMV). Postnatally, Ob-R and Ob-Rb mRNA begins to be observed in the
dorsomedial nucleus (DMH). Relativevalues are given as qualitative estimates of the expression of Ob-R and Ob-Rb mRNA. A three-point scalewas used to rate the
data: +, moderate to high density; +/?, low density; ?, no signal. The data are based on analysis of autoradiographs of representative animals.
Fig. 5. Quantification of [125I]leptin binding in the choroid plexus of the lateral
ventricle and of the dorsal third ventricle and in the ependymal layer of the third
ventricle. Values were converted to percentages of E18 values (100%). Data are
presented as means ? S.E.M. Significant differences between developmental
stagesareindicatedas**P < 0.01and***P < 0.001.Animalnumbersinbrackets.
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isoforms of the leptin receptor and [125I]leptin binding were
detected in the choroid plexus and in the leptomeninges at all
stages studied. Quantification of [125I]leptin binding showed an
increase of the level of binding to both the choroid plexus of the
lateral ventricle and the third dorsal ventricle throughout
development up to P3. These results indicate an increase of
leptin receptors in the choroid plexus during development.
Farra et al., 2000). A possible explanation for this lack of leptin
binding is that most of the leptin receptor in neurones of the
hypothalamus is found in the cytoplasm, especially in the Golgi
apparatus, rather than at the cell surface, as demonstrated by
immunocytochemistry (De Matteis and Cinti, 1998; Diano
et al., 1998; Baskin et al., 1999b). Consistent with these
findings, low levels of Ob-Rb expression are detected at the cell
surface of transiently transfected HeLa cells (Belouzard et al.,
2004). It is also conceivable that Ob-R mRNA is not translated
intoreceptorproteinatthe earlier stagesofdevelopmentlooked
at in the present study.
Unlike adult animals (Mercer et al., 1996; Fei et al., 1997),
Ob-Rb mRNA was highly expressed in the ependymal layer of
the third ventricle at E18, E21 and P3. There was also relatively
strong [125I]leptin binding in this area, which is absent in adult
animals (Baskin et al., 1999a). [125I]leptin binding to this region
was present at each stage studied. Leptin gene expression in the
ependymal layer of the third ventricle has previously been
reported in postnatal day 2 rats (Morash et al., 2001) and Ob-Rb
immunoreactivity has been reported in the ventricular layer at
E14, E18 and P0 (Matsuda et al., 1999). It is known that the
ependymal layer of the third ventricle consists of ciliated and
non-ciliated cells without distinct subventricular cell layers.
Some non-ciliated cells are called tanycytes and have long cell
cells were reported to be multipotent and to generate
hypothalamic neurones (De Vitry et al., 1980). Since the
ventricular layercontainsprematureneuronalcells and is a zone
where neurogenesis occurs (De Vitry et al., 1980; Xu et al.,
role of leptin in the development of the hypothalamus. Ob-Rb
may be transiently expressed in the ependymal layer of the third
ventricle in cells that subsequently migrate to hypothalamic
inthe quantityof[125I]leptinbinding tothisareawith increasing
developmental age. Most of the hypothalamic neurones are in
place at E16 as peak birthdates of hypothalamic nuclei occur
around E12/E13 for the lateral zone, around E14/E15 for the
medial zone (for example in the ARC) and around E16/E17 for
the periventricular zone (Ifft, 1972; Altman and Bayer, 1986).
However, neurones occupying the anteromedial and poster-
omedial positions settle during the latter stage of embryonic life
the median eminence becomes progressively differentiated
within the first 2 weeks after birth in the rat (for review, see
(Tixier-Vidal and De Vitry, 1979).
Consistent with previous work in the embryonic and adult
brain (Devosetal., 1996; MalikandYoung, 1996; Mercer etal.,
1996; Hoggard et al., 1997; Dal Farra et al., 2000), short
It was initially thought that short leptin receptor isoforms
may transport leptin from the peripheral circulation into the
cerebrospinal fluid (CSF) and the CNS (Tartaglia et al., 1995;
Bjorbaek et al., 1998; Hileman et al., 2002). However not all
studies support this hypothesis. For instance, leptin is still
present in the CSF of Koletsky rats (Banks et al., 2002), which
lack any leptin receptor isoform due to a mutation in the
extracellular domain of the receptor (Wu-Peng et al., 1997).
This finding indicates that the brain blood-barrier (BBB)
transporter of leptin is not encoded by Ob-R and the role of
leptin receptors in the choroid plexus remains unclear.
Leptin targets also include cranial nerves. It has been
reported that neuronal size in the motor nucleus of the facial
nerve was greatly decreased in ob/ob mice (Bereiter and
Jeanrenaud, 1979) and also that Ob-Rb mRNA is expressed in
the motor nucleus of the facial nerve in mouse from embryonic
day 16.5 (Udagawa et al., 2000). In addition, db/db mice were
shown to have greater gustatory neural sensitivities (Ninomiya
et al., 1995, 1998; Sako et al., 1996) and higher behavioural
preferences for various sweet substances (Ninomiya et al.,
1995) compared with lean control mice. Taken together these
results suggest that leptin has an important role in the growth of
the facial nucleus and also that leptin may be a modulator of
taste. In support of this role for leptin, the short isoforms of the
leptin receptor were found to be expressed in the trigeminal
confirmed by leptin receptor autoradiography with [125I]leptin
binding. Trigeminal ganglion neurones innervate mainly
mechanoreceptors, thermoreceptors and nociceptors in the
face, oral cavity and nasal cavity (Dubner, 1978; Davies,1988).
Notably, the innervation of the anterior two-thirds of the tongue
is innervated by mandibulary nerve, one of the three major
divisions of the trigeminal nerve (Martin, 1988). Textural
characteristics and temperature of foods are thus detected by
trigeminal mechano and thermal sensors on the tongue and
throughout the entire oral cavity (for review, see (Berthoud,
2002)). As well as Ob-R gene expression in the trigeminal
ganglion, orexin positive immunoreactive fibers and melanin-
concentrating hormone receptors are observed in the motor
observations support the hypothesis that leptin could influence
feeding behaviour by modulating the trigeminal nerve.
of neuronal leptin receptors commencing with the ARC. In
addition, the sequential increase in the level of [125I]leptin
binding in the choroid plexus compared to a decrease in
[125I]leptin binding to the ependymal layer of the third ventricle
on region in embryonic and neonatal brain.
We wish to thank Elke Thom and Elvira Stromeyer for their
excellent technical help, Pat Bain for assistance with graphics
and Dr. N. Hoggard, Rowett Research Institute for kindly
providing the Ob-R and Ob-Rb riboprobes.
A.-S. Carlo et al./Journal of Chemical Neuroanatomy 33 (2007) 155–163 161
Author's personal copy
Chua Jr., S.C., Koutras, I.K., Han, L., Liu, S.M., Kay, J., Young, S.J., Chung,
W.K., Leibel, R.L., 1997. Fine structure of the murine leptin receptor gene:
splice site suppression is required to form two alternatively spliced tran-
scripts. Genomics 45, 264–270.
J.R., Wilson, S., Buckingham, R.E., Evans, M.L., Leslie, R.A., Williams,
G., 1999. Differential distribution of orexin-A and orexin-B immunoreac-
tivity in the rat brain and spinal cord. Peptides 20, 1455–1470.
Grant support: Anne-Sophie Carlo was a recipient of a
Research Training Grant at Obesechool funded by EC
framework V Program: HPMT-CT-2001-00410 and NuGo
Exchange Grant No. DS05-007 funded by EC framework VI
Ahima, R.S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Maratos-Flier,
Nature 382, 250–252.
Altman, J., Bayer, S.A., 1986. The development of the rat hypothalamus. Adv.
Anat. Embryol. Cell Biol. 100, 1–178.
Banks, W.A., Niehoff, M.L., Martin, D., Farrell, C.L., 2002. Leptin transport
across the blood-brain barrier of the Koletsky rat is not mediated by a
product of the leptin receptor gene. Brain Res. 950, 130–136.
Baskin, D.G., Breininger, J.F., Bonigut, S., Miller, M.A., 1999a. Leptin
binding in the arcuate nucleus is increased during fasting. Brain Res.
Baskin, D.G., Schwartz, M.W., Seeley, R.J., Woods, S.C., Porte Jr., D.,
Breininger, J.F., Jonak, Z., Schaefer, J., Krouse, M., Burghardt, C., Camp-
field, L.A., Burn, P., Kochan, J.P., 1999b. Leptin receptor long-form splice-
variant protein expression in neuron cell bodies of the brain and co-
localization with neuropeptide Y mRNA in the arcuate nucleus. J. Histo-
chem. Cytochem. 47, 353–362.
Baumann, H., Morella, K.K., White, D.W., Dembski, M., Bailon, P.S., Kim, H.,
Lai, C.F.,Tartaglia, L.A.,1996. Thefull-length leptinreceptorhassignaling
capabilities of interleukin 6-type cytokine receptors. Proc. Natl. Acad. Sci.
U.S.A. 93, 8374–8378.
receptor at the cell surface result from constitutive endocytosis and intra-
cellular retention in the biosynthetic pathway. J. Biol. Chem. 279, 28499–
Bereiter, D.A., Jeanrenaud, B., 1979. Altered neuroanatomical organization in
the central nervous system of the genetically obese (ob/ob) mouse. Brain
Res. 165, 249–260.
Bereiter, D.A., Jeanrenaud, B., 1980. Altered dendritic orientation of hypotha-
lamic neurons from genetically obese (ob/ob) mice. Brain Res. 202, 201–
weight. Neurosci. Biobehav. Rev. 26, 393–428.
Bjorbaek, C., Elmquist, J.K., Michl, P., Ahima, R.S., van Bueren, A., McCall,
A.L., Flier, J.S., 1998. Expression of leptin receptor isoforms in rat brain
microvessels. Endocrinology 139, 3485–3491.
Bouret, S.G., Draper, S.J., Simerly, R.B., 2004a. Formation of projection
pathways from the arcuate nucleus of the hypothalamus to hypothalamic
regions implicated in the neural control of feeding behavior in mice. J.
Neurosci. 24, 2797–2805.
Bouret, S.G., Draper, S.J., Simerly, R.B., 2004b. Trophic action of leptin on
hypothalamic neurons that regulate feeding. Science 304, 108–110.
Buyse, M., Ovesjo, M.L., Goiot, H., Guilmeau, S., Peranzi, G., Moizo, L.,
Walker, F., Lewin, M.J., Meister, B., Bado, A., 2001. Expression and
regulation of leptin receptor proteins in afferent and efferent neurons of
the vagus nerve. Eur. J. Neurosci. 14, 64–72.
a Golgi study. Am. J. Anat. 151, 173–189.
Casanueva, F.F., Dieguez, C., 1999. Neuroendocrine regulation and actions of
leptin. Front Neuroendocrinol. 20, 317–363.
monoiodoleptin analog to mouse tissues: a developmental study. Peptides
Davies, A.M., 1988. The trigeminal system: an advantageous experimental
model for studying neuronaldevelopment. Development 103 (Suppl.),175–
De Matteis, R., Cinti, S., 1998. Ultrastructural immunolocalization of leptin
receptor in mouse brain. Neuroendocrinology 68, 412–419.
De Vitry, F., Picart, R., Jacque, C., Legault, L., Dupouey, P., Tixier-Vidal, A.,
1980. Presumptivecommon precursor for neuronaland glial cell lineages in
mouse hypothalamus. Proc. Natl. Acad. Sci. U.S.A. 77, 4165–4169.
Devos, R., Richards, J.G., Campfield, L.A., Tartaglia, L.A., Guisez, Y., van der
Heyden, J., Travernier, J., Plaetinck, G., Burn, P., 1996. OB protein binds
specifically to the choroid plexus of mice and rats. Proc. Natl. Acad. Sci.
U.S.A. 93, 5668–5673.
Diano, S., Kalra, S.P., Horvath, T.L., 1998. Leptin receptor immunoreactivity is
associatedwiththe Golgiapparatusofhypothalamic neuronsand glialcells.
J. Neuroendocrinol. 10, 647–650.
Dubner, R., 1978. Neurophysiology of pain. Dent. Clin. North Am. 22, 11–30.
Elmquist, J.K., Bjorbaek, C., Ahima, R.S., Flier, J.S., Saper, C.B., 1998a.
Distributions of leptin receptor mRNA isoforms in the rat brain. J. Comp.
Neurol. 395, 535–547.
Elmquist, J.K., Maratos-Flier, E., Saper, C.B., Flier, J.S., 1998b. Unraveling the
central nervous system pathways underlying responses to leptin. Nat.
Neurosci. 1, 445–450.
Fei, H., Okano, H.J., Li, C., Lee, G.H., Zhao, C., Darnell, R., Friedman, J.M.,
1997. Anatomiclocalization ofalternativelyspliced leptinreceptors(Ob-R)
in mouse brain and other tissues. Proc. Natl. Acad. Sci. U.S.A. 94, 7001–
Garris, D.R.,1989.Morphometricanalysis of obesity (ob/ob)-and diabetes(db/
db)-associated hypothalamic neuronal degeneration in C57BL/KsJ mice.
Brain Res. 501, 162–170.
Ge, H., Huang, L., Pourbahrami, T., Li, C., 2002. Generation of soluble leptin
receptor by ectodomain shedding of membrane-spanning receptors in vitro
and in vivo. J. Biol. Chem. 277, 45898–45903.
Hileman, S.M., Pierroz, D.D., Masuzaki, H., Bjorbaek, C., El-Haschimi, K.,
Banks, W.A., Flier, J.S., 2002. Characterizaton of short isoforms of the
leptin receptor in rat cerebral microvessels and of brain uptake of leptin in
mouse models of obesity. Endocrinology 143, 775–783.
Hoggard, N., Hunter, L., Duncan, J.S., Williams, L.M., Trayhurn, P., Mercer,
J.G., 1997. Leptin and leptin receptor mRNA and protein expression in the
murine fetus and placenta. Proc. Natl. Acad. Sci. U.S.A. 94, 11073–11078.
Hoggard, N., Hunter, L., Trayhurn, P., Williams, L.M., Mercer, J.G., 1998.
Leptin and reproduction. Proc. Nutr. Soc. 57, 421–427.
Ifft, J.D., 1972. An autoradiographic study of the time of final division of
neurons in rat hypothalamic nuclei. J. Comp. Neurol. 144, 193–204.
Lynn, R.B., Cao, G.Y., Considine, R.V., Hyde, T.M., Caro, J.F., 1996. Auto-
radiographic localization of leptin binding in the choroid plexus of ob/ob
and db/db mice. Biochem. Biophys. Res. Commun. 219, 884–889.
Malik, K.F., Young 3rd, W.S., 1996. Localization of binding sites in the central
nervous system for leptin (OB protein) in normal, obese (ob/ob), and
diabetic (db/db) C57BL/6J mice. Endocrinology 137, 1497–1500.
Martin, J.H., 1988. General organization of the cranial nerve nuclei and the
Appleton & Lange, pp. 301–320.
Matsuda, J., Yokota, I., Tsuruo, Y., Murakami, T., Ishimura, K., Shima, K.,
Kuroda, Y., 1999. Development changes in long-form leptin receptor
expression and localization in rat brain. Endocrinology 140, 5233–
Mercer, J.G., Hoggard, N., Williams, L.M., Lawrence, C.B., Hannah, L.T.,
Trayhurn,P.,1996.LocalizationofleptinreceptormRNAand thelong form
splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions
by in situ hybridisation. FEBS Lett. 387, 113–116.
Mercer, J.G., Moar, K.M., Hoggard, N., 1998. Localization of leptin receptor
(Ob-R) messenger ribonucleic acid in the rodent hindbrain. Endocrinology
Millhouse, O.E., 1971. A Golgi study of third ventricle tanycytes in the adult
rodent brain. Z. Zellforsch. Mikrosk. Anat. 121, 1–13.
A.-S. Carlo et al./Journal of Chemical Neuroanatomy 33 (2007) 155–163162
Author's personal copy
Morash, B., Wilkinson, D., Murphy, P., Ur, E., Wilkinson, M., 2001. Devel-
Cell. Endocrinol. 185, 151–159.
Murray, J.F., Mercer, J.G., Adan, R.A., Datta, J.J., Aldairy, C., Moar, K.M.,
Baker, B.I., Stock, M.J., Wilson, C.A., 2000. The effect of leptin on
luteinizing hormone release is exerted in the zona incerta and mediated
by melanin-concentrating hormone. J. Neuroendocrinol. 12, 1133–
Niimi, K., Fujiwara, N., Takimoto, T., Matsugi, S., 1962. The course and
termination of the ascending fibers of the brachium conjunctivum in the cat
as studied by the Nauta method. Tokushima J. Exp. Med. 8, 269–284.
Ninomiya, Y., Imoto, T., Yatabe, A., Kawamura, S., Nakashima, K., Katsu-
kawa, H., 1998. Enhanced responses of the chorda tympani nerve to
nonsugar sweeteners in the diabetic db/db mouse. Am. J. Physiol. 274,
Ninomiya, Y., Sako, N., Imai, Y., 1995. Enhanced gustatory neural responses to
sugars in the diabetic db/db mouse. Am. J. Physiol. 269, R930–R937.
Paxinos, G., Ashwell, K.W.S., To ¨rk, I., 1994. Atlas of the Developing Rat
Nervous System, second ed. London Academic Press, Inc..
Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates,
second ed. London Academic Press, Inc..
Saito, Y., Cheng, M., Leslie, F.M., Civelli, O., 2001. Expression of the melanin-
concentrating hormone (MCH) receptor mRNA in the rat brain. J. Comp.
Neurol. 435, 26–40.
Sako, N., Ninomiya, Y., Fukami, Y., 1996. Analysis of concentration-response
relationship for enhanced sugar responses of the chorda tympani nerve in
the diabetic db/db mouse. Chem. Senses 21, 59–63.
Schwartz, M.W., Woods, S.C., Porte Jr., D., Seeley, R.J., Baskin DG, 2000.
Central nervous system control of food intake. Nature 404, 661–671.
Sena, A., Sarlieve, L.L., Rebel, G., 1985. Brain myelin of genetically obese
mice. J. Neurol. Sci. 68, 233–243.
Shimada, M., Nakamura, T., 1973. Time of neuron origin in mouse hypotha-
lamic nuclei. Exp. Neurol. 41, 163–173.
Steppan, C.M., Swick, A.G., 1999. A role for leptin in brain development.
Biochem. Biophys. Res. Commun. 256, 600–602.
Tartaglia, L.A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R.,
tion and expression cloning of a leptin receptor, OB-R. Cell 83, 1263–1271.
Tixier-Vidal, A., De Vitry, F., 1979. Hypothalamic neurons in cell culture. Int.
Rev. Cytol. 58, 291–331.
leptin receptor (Ob-Rb) mRNA in the brain of mouse embryos and newborn
mice. Brain Res. 868, 251–258.
Waelput, W., Brouckaert, P., Broekaert, D., Tavernier J, 2006. A role for leptin
in the systemic inflammatory response syndrome (SIRS) and in immune
response, an update. Curr. Med. Chem. 13, 465–475.
the extracellular domain of the leptin receptor (Lepr): evidence for deficient
Diabetes 46, 513–518.
Xu, Y., Tamamaki, N., Noda, T., Kimura, K., Itokazu, Y., Matsumoto, N.,
rat 3rd ventricle. Exp. Neurol. 192, 251–264.
A.-S. Carlo et al./Journal of Chemical Neuroanatomy 33 (2007) 155–163163