A sensitive period for environmental regulation of
eating behavior and leptin sensitivity
Marco Mainardia,1, Gaia Scabiab,c,1, Teresa Vottarib,c,1, Ferruccio Santinic, Aldo Pincherac, Lamberto Maffeia,d,
Tommaso Pizzorussod,e,2, and Margherita Maffeib,c,f,2
aLaboratory of Neurobiology, Scuola Normale Superiore,bDulbecco Telethon Institute, andcDepartment of Endocrinology and Kidney, University-Hospital of
Pisa,dNeuroscience Institute, National Research Council, 56100 Pisa, Italy;eDepartment of Psychology, University of Florence, 50139 Florence, Italy; and
fInstitute of Food Sciences, National Research Council, 83100 Avellino, Italy
Edited* by Jeffrey M. Friedman, The Rockefeller University, New York, NY, and approved August 10, 2010 (received for review October 14, 2009)
Western lifestyle contributes to body weight dysregulation. Leptin
down-regulates food intake by modulating the activity of neural
circuits in the hypothalamic arcuate nucleus (ARC), and resistance
to this hormone constitutes a permissive condition for obesity.
Physical exercise modulates leptin sensitivity in diet-induced obese
rats. The role of other lifestyle components in modulating leptin
sensitivity remains elusive. Environmentally enriched mice were
used to explore the effects of lifestyle change on leptin production/
action and other metabolic parameters. We analyzed adult mice
exposed to environmental enrichment (EE), which showed de-
creased leptin, reduced adipose mass, and increased food intake.
exercise (YW) since birth, both of which showed decreased leptin.
YEE mice showed no change in food intake, increased response to
leptin administration, increased activation of STAT3 in the ARC. The
YWleptin-induced food intake responsewas intermediatebetween
young mice kept in standard conditions and YEE. YEE exhibited
increased and decreased ratios of excitatory/inhibitory synapses
neurons of the ARC, respectively. We also analyzed animals as de-
scribed for YEE and then placed in standard cages for 1 mo. They
showed no altered leptin production/action but demonstrated
changes in excitatory/inhibitory synaptic contacts in the ARC similar
toYEE. EEandphysical activity resultedin improved insulin sensitiv-
ity. In conclusion, EE and physical activity had an impact on feeding
behavior, leptin production/action, and insulin sensitivity, and EE
environmental enrichment|arcuate nucleus|AgRP|POMC|synaptic
societies characterized by limited physical activity, excessive ca-
loric intake, and repetitive behavioral patterns contributes to the
dysregulation of the otherwise homeostatic control of body weight
(BW) (1). The main player in this system is leptin, a hormone se-
creted in the periphery by fat cells (2), which signals the status of
energy expenditure by activating signal transduction mediated by
the JAK-STAT pathway in the hypothalamic arcuate nucleus
(ARC) through its receptor (Ob-Rb). This, in turn, promotes ex-
citation and inhibition ofneurons expressing, respectively, POMC,
the most potent anorexigenic peptide, and the orexigenic peptides
agouti-related peptide (AgRP)/Neuropeptide Y (NPY) (3, 4).
Such a seemingly clear view of the complex regulation of
feeding behavior and BW is challenged by the fact that the ma-
jority of obese people exhibit high levels of circulating leptin (5),
to which they are apparently resistant. Leptin resistance is
emerging as a permissive condition for obesity (6), and efforts to
enhance leptin sensitivity could be a determinant in the treatment
and prevention of this disorder.
t is widelyacceptedthattheprevalentlifestylemodelofWestern
Leptin resistance, often reported in standard housed mice (7),
or physical exercise (8). However, mice used in the studies in-
vestigating leptin regulation and action are usually reared under
standard conditions that allow little physical activity and limited
sensorial, emotional, and cognitive stimulation. This certainly
impinges on several metabolic functions, as Martin et al. (9) con-
clude from a metaanalysis of health conditions of standard housed
mice vs. physically exercised or diet-restricted mice. What is not as
yet clear is whether rearing factors other than diet or locomotor
activity may affect metabolism in rodents, as is indicated by some
evidence in humans. Indeed, depression, anxiety, solitude, frustra-
tion, and boredom are considered important determinants of hu-
In the present study, we asked whether multifaceted mod-
ifications of the environment might influence metabolism and
leptin sensitivity in WT nonobese animals. To address this issue,
we exposed mice to environmental enrichment (EE), a manipu-
lation of the rearing environment that includes enhanced physical
activity and sensory, cognitive, and social stimulation (12) and
exerts important effects on experience-dependent plasticity, in-
cluding adult neurogenesis and synaptic connectivity (12). To
disentangle the contribution of locomotor activity from the other
EE components, the effects of EE were compared with those of
voluntary physical exercise alone.
Our findings demonstrate that EE has an effect on glucose tol-
erance, feeding behavior, and leptin sensitivity; this third aspect is
observed only if EE has been applied since birth. In the ARC of
young mice, these effects are associated with increased leptin re-
ceptor expression, enhanced activation of the STAT3 pathways,
α-MSH neurons and toward the latter in AgRP/NPY neurons. In-
triguingly, physical activity also had an impact on feeding behavior
and leptin production/action and EE affected ARC circuitry.
EE Effects on Metabolism and Feeding Behavior in Adult Mice. Ex-
posure of male adult mice [postnatal day (P) 50] to EE for 4 wk
resulted in no significant difference in BW (Table S1). A signifi-
cant difference was seen in the weight of the fat depots, with adult
mice raised under the EE condition (AEE) showing significantly
lower values for this parameter as compared with adult mice
reared under standard conditions (AST) (Table S1). No differ-
Author contributions: F.S., L.M., T.P., and M. Maffei designed research; M. Mainardi, G.S.,
and T.V. performed research; M. Mainardi, G.S., and T.V. analyzed data; and A.P., L.M.,
T.P., and M. Maffei wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1M. Mainardi, G.S., and T.V. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 21, 2010
| vol. 107
| no. 38
ence was found in the weight of other metabolically active organs,
including the liver and brown adipose tissue (BAT) (Table S1).
lower in AEE mice as compared with AST mice (Fig. 1A). When
the individual leptin levels at 4 wk were normalized for the cor-
responding weight of the fat pads, no significant difference be-
tween the AEE and AST mice could be observed (Table S1),
suggesting that the reduction in serum leptin likely results from
the decrease in adiposity herein described for the EE mice. When
leptin mRNA abundance was assessed in the epididymal white
adipose tissue (WAT) of AEE and AST mice, no significant dif-
ference was found (Fig. 1B).
Lowering the levels of circulating leptin may result in increased
food intake. Indeed, average food intake was significantly higher
(Table S1) in the AEE mice, as expected in an animal exposed to
a higher level of physical activity.
AEE mice showed similar insulin levels and moderately de-
creased levels of fasting glucose as compared with AST mice
(Table S1). When an i.p. glucose tolerance test (IpGTT) was
observed (Fig. 1D).
Taken together, these data indicate that EE improves glucose
tolerance, reduces adipose mass, increases food intake, and
down-regulates leptin production in the adult mouse.
EE Effects on Metabolism and Feeding Behavior in Young Mice. To
leptin production/action, we monitored a group of young mice
born under the EE condition (YEE) and killed at P50. Young
control mice were kept in standard conditions (YST). The BW
and weight of the liver and BAT were identical in YEE and YST
mice. No difference was seen in the weight of the gonadal fat pad
YEE mice showed a similar plasma insulin level and a slightly
lower level of fasting glucose (Table S2) and performed better
during the IpGTT (Fig. 2 A and B). Plasma leptin was lower in
YEE mice than in YST mice, even when normalized to the weight
of fat pads(Fig. 3 A and B and Table S2). The abundance of WAT
and YST mice, but no statistically significant difference was found
(Fig. 3C). Other posttranscriptionalmechanisms could explain the
different leptin levels in YEE mice. For instance, Ceccarini et al.
(13) reported that leptin uptake by megalin in the kidney and its
binding to red bone marrow importantly accounts for its bio-
distribution and may contribute to explain variation in its plasma
levels. The diminished level of circulating leptin observed in YEE
mice was not associated with an increase in food intake (Fig. 3D).
These data indicate that EE has a positive effect on glucose
tolerance, no effect on adipose mass or food intake, and reduces
leptin production during development.
EE Effects on Leptin Response and Hypothalamic Gene Expression in
Young Mice. We then analyzed the effect of leptin administration
on food consumption. Food intake assessed 14 h after injection of
leptin or vehicle was reduced by 38% in YEE mice and by 13% in
YST mice (as compared with corresponding vehicle-treated ani-
mals) (Fig. 4A). The relatively small reduction of food intake
measured in YST mice should not be surprising, given that leptin
effects in WT animals on food intake are often cumulative and
become significant only after 2–3 d of chronic treatment (14). The
long-isoform leptin receptor (Ob-Rb), the orexigenic peptide
NPY, the α-MSH precursor POMC, and orexin. Real-time PCR
revealed no significant difference in the abundance of POMC and
NPY transcripts between YEE and YST mice, although Ob-Rb
and orexin expression was up-regulated in YEE mice (Fig. 4B).
Next, we asked whether the activation state of molecules in-
volved in leptin signaling might differ between the two groups. On
leptin injection, the number of phospho-STAT3 (pSTAT3)-
4C). In particular, leptin stimulation resulted in STAT3 phos-
saline-treated mice), whereas pSTAT3 was activated in only 38%
of STAT3-positive neurons in YST mice (190% of saline-treated
mice). No significant difference was found in the number of neu-
rons expressing STAT3 in YEE and YST mice (Fig. 4D).
These data are consistent with the above-stated hypothesis
that EE increases leptin sensitivity in young mice.
EE Effects on ARC Synaptic Connectivity in Young Mice. Considering
that fasting and genetic leptin deficiency also affect energy bal-
ance by significantly interfering with the synaptic plasticity of the
ARC (4, 15, 16), we analyzed whether EE has an impact on ARC
In the cerebral cortex and hippocampus (17), EE modifies
synaptic organization of neural circuits and enhances the ex-
pression of BDNF (18), a neurotrophin important for synaptic
plasticity.When hypothalamicBDNFexpression wasassessed,we
Plasma leptin at the end of EE in AST (n = 16) and AEE (n = 14) mice (t test, *P <
0.05). (B) WAT leptin gene expression in AST (n = 11) and AEE (n = 10) mice. (C)
IpGTT results at the end of EE in AST (n = 11) and AEE (n = 10) mice (two-way
for the glycemic responses of AST and AEE mice shown in C (t test, ***P < 0.001).
EffectofEEonleptinexpressionandglucosetolerancein adult mice.(A)
results at theendofEE inYST (n = 19)and YEE(n = 15)mice (two-wayANOVA:
rearing effect, P < 0.001; time effect, P < 0.001.Bonferroni post hoc test in YST
vs. YEE mice, *P < 0.05; ***P < 0.001). (B) AUC values (mg·dL·min−1over
a 120-min test) for the glycemic responses of YST and YEE mice shown in A
(t test, **P < 0.002).
Improved glucose tolerance in young mice at the end of EE. (A) IpGTT
| www.pnas.org/cgi/doi/10.1073/pnas.0911832107 Mainardi et al.
found a significantly increased level in YEE mice as compared
with YST mice (Fig. 5A).
We then searched for modifications in the neural circuitry of
the ARC. To this end, we assessed the number of excitatory and
inhibitory synapses in YEE and YST mice by immunofluores-
cence, counting the puncta labeled with the specific markers
vGluT2 and vGAT, corresponding to excitatory and inhibitory
synaptic terminals, respectively. We found that in the ARC of
YEE mice, the number of excitatory synaptic terminals is signif-
icantly higher than in YST mice and the number of inhibitory
synaptic terminals is reduced (Fig. S1 A and B), thus lowering the
ratio between excitatory and inhibitory synapses (Fig. S1C).
We next asked whether excitatory and inhibitory synapses on
neurons expressing α-MSH and AgRP were differently affected by
vGAT. As shown in Fig. 5B, the ratio of vGluT2/vGAT-positive
puncta is significantly higher on α-MSH-positive neurons of YEE
mice as compared with YST mice. On the other hand, YEE mice
exhibit a significantly lower ratio of vGluT2/vGAT-positive puncta
on AgRP neurons (Fig. 5C). The YEE effects observed with the
puncta in each of the four double-immunofluorescence experi-
ments shown in Fig. S2.
These data establish that on EE in young mice, the ARC
undergoes a change in its overall synaptic connectivity and a cell-
specific alteration in neurons that are key in the regulation of
How EE Experienced During Development Affects Metabolism and
Feeding Behavior in the Adult Mouse. We next asked whether EE
effects could persist once animals are removed from this condi-
tion. Mice enriched since birth were transferred to a standard
cage at P50 and monitored until P80 (EE/ST). A group of control
mice (ST/ST) was kept in standard cages during the whole period.
Despite displaying a similar BW at P50, the two groups diverged
afterward in that EE/ST mice showed a greater weight gain be-
tween P50 and P80, ending up with a significantly greater weight as
intheweightofthe epigonadalfat,BAT,orliver(Table S3).ST/ST
(Table S3). IpGTT results and area under the curve (AUC) cal-
culations revealed significantly improved glucose tolerance in EE/
ST mice as compared with ST/ST mice (Fig. 6 A and B).
The increased BW observed in EE/ST mice can be explained,
considering that they ate significantly more between P50 and P80
(Fig. 6C). Interestingly, food intake increased abruptly after ani-
mal transfer to standard rearing. This sharp change in food con-
sumption was associated with a concomitant rise in leptin levels
that increased 3 d after the transition from the EE condition to
standard conditions (P53) with respect to the levels observed 1 d
before the end of the EE condition (P49) (Fig. 6D). ST/ST mice
did not show significantly increased leptin over this time window.
is decreased in YEE (n = 37) compared with YST (n = 34) mice (t test, *P < 0.05).
(B) Plasma leptin/epididymal (epi) fat weight (pg·mL·mg−1) for YST and YEE
mice (t test, **P < 0.01). (C) Leptin gene expression in epididymal WAT of YST
(n = 19) and YEE (n = 17) mice. (D) Average food intake in YST and YEE mice.
Effect of EE on metabolic parameters in young mice. (A) Plasma leptin
istration in young mice. (A) Food intake assessed 14 h after
i.p. leptin injection in YST and YEE mice [two-way ANOVA:
treatment effect, P < 0.01; interaction, P < 0.05. Bonferroni
post hoc test in YST vs. YEE mice, *P < 0.05; in saline (sal) vs.
leptin (lep),§§§P < 0.001]. (B) Hypothalamic gene expression
in YST (n = 15) and YEE (n = 13) mice (t test, *P < 0.05). (C)
(Left) Histogram showing the ratio of pSTAT3-positive cells
to the total number of STAT3-positive neurons 45 min after
leptin or saline injection in the ARC of YST (saline, n = 8;
leptin, n = 6) and YEE (saline, n = 7; leptin, n = 6) mice (two-
way ANOVA: rearing effect, P < 0.001; treatment effect, P <
0.001; interaction, P = 0.037. Bonferroni post hoc tests in YST
vs. YEE mice, ***P < 0.001; in leptin vs. saline,§P < 0.05;
§§§P < 0.001). (Right) Representative immunofluorescence
showing pSTAT3 activation in the ARC after saline or leptin
injection. (Scale bar: 100 μm.) (D) (Left) Total number of
STAT3-positive neurons in the ARC of YEE and YST mice
after saline or leptin injection (two-way ANOVA, NS, not
significant). (Right) Representative immunofluorescence
showing STAT3 expression in the ARC of YEE and YST mice.
(Scale bar: 100 μm.)
EE enhances response to exogenous leptin admin-
Mainardi et al.PNAS
| September 21, 2010
| vol. 107
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At P80,there was no difference in the plasma leptin level between
EE/ST and ST/ST mice (Table S3).
Taken together, these data indicate that EE experienced
during developmental age leaves a metabolic imprint on the
adult mouse, which shows enhanced glucose tolerance.
How EE Experienced During Development Affects Leptin Response in
the Adult Mouse. Food intake measured 14 h following leptin
injection was not significantly different in P80 EE/ST and ST/ST
mice (Fig. S3A). The assessment of leptin sensitivity in terms of
STAT3 activation following leptin injection mirrored this sce-
nario: EE/ST and ST/ST mice did not exhibit a significant dif-
ference in the percentage of STAT3-positive neurons showing
staining for pSTAT3 (Fig. S3B). No significant difference was
found in the number of neurons expressing STAT3 in EE/ST and
ST/ST mice (Fig. S3C).
Our data show that the effects of EE on leptin sensitivity ob-
served in developing young animals do not persist through
adulthood on standard rearing conditions.
How EE Experienced During Development Affects ARC Synaptic
Connectivity in the Adult Mouse. EE/ST mice showed a signifi-
cantly lower number of inhibitory terminals (vGAT-positive) in
the ARC with respect to the ST/ST condition (Fig. S4B). No
difference was found in the number of excitatory synaptic termi-
nals (vGluT2-positive) (Fig. S4A). The ratio between excitatory
and inhibitory puncta was significantly higher in EE/ST mice as
compared with ST/ST mice (Fig. S4C).
When neuron-specificinvestigations wereperformed, wefound
that only the reduction in vGAT puncta on α-MSH neurons and
the increase in vGluT2 puncta on AgRP-positive neurons were
significant in EE/ST mice as compared with ST/ST mice (Fig. S5
A–D).Nonetheless, the excitation/inhibition ratiofor EE/ST mice
was respectively higher in α-MSH neurons (Fig. 7A) and lower in
AgRP neurons (Fig. 7B). Hypothalamic BDNF gene expression
was similar in EE/ST and ST/ST mice (0.63 ± 0.14 and 0.55 ± 0.05
arbitrary units, respectively).
These results indicate that ARC circuits retain a persistent,
although attenuated, trace of the rearing condition experienced
during development consisting of an altered ratio between ex-
citatory and inhibitory synaptic density affecting both α-MSH
and AgRP neurons.
Comparison Between Physical Exercise and EE. To assess whether
the effects of EE on feeding behavior, leptin production and re-
sponsiveness, glucose tolerance, and synaptic organization of the
ARCin youngmice weresimply attributable toenhanced physical
activity, we studied how voluntary physical exercise (free access to
a running wheel) by itself could influence these parameters.
Young physically exercised (YW) mice did not show a signifi-
a trend toward increased food intake (Fig. S6A). Their adiposity
(weight of epididymal fat pad/BW) was lower than that observed
in both YST and YEE mice (Fig. S6B). Results of the IpGTT
revealed that, similar to YEE mice, YW mice displayed better
glucose tolerance as compared with YST mice (Fig. S6 D and E).
YW mice showed a lower leptin concentration than YST mice
injection, was intermediate between that of YST mice and that of
YEE mice. If we assume that the leptin-induced change in food in-
leptin injection, the ratio between pSTAT3 and STAT3-positive
that in YEE mice. However, if the fold change from saline to leptin
treatment is considered, this parameter underwent a 2.55-fold
that observed in YEE (2.34) and YST (1.9) mice (Fig. S6G).
Different from what was observed with EE, no effect of vol-
untary physical exercise was observed on the total number of
excitatory and inhibitory synapses in the ARC (Fig. S6H).
Taken together, these data suggest that physical exercise by
itself quantitatively accounts for only some of the changes in
metabolism and leptin sensitivity observed on EE.
neurons of YST and YEE mice. (A) BDNF hypothalamic expression in YST (n =
12) and YEE (n = 7) mice (t test, **P < 0.01). (B) Histogram of the ratio be-
tween excitatory and inhibitory synapses on α-MSH neurons in the ARC of
YEE (n = 11) and YST (n = 9) mice (t test, ***P < 0.001). (C) Histogram of the
ratio between excitatory and inhibitory synapses on AgRP neurons in the
ARC of YEE (n = 7) and YST (n = 6) mice (t test, *P < 0.05).
BDNF expression and excitation/inhibition ratio on α-MSH and AgRP
parameters in adulthood. (A) IpGTT results in ST/ST (n = 13) and EE/ST (n = 19)
mice (two-way ANOVA: rearing effect, P < 0.0001; time effect, P < 0.0001;
interaction, P < 0.0001. Bonferroni post hoc test in ST/ST vs. EE/ST, *P < 0.05;
***P < 0.001). (B) AUC values (mg·dL·min−1over a 120-min test) for the gly-
cemic responses of ST/ST and EE/ST mice shown in A (t test, ***P < 0.001). (C)
Weekly food intake of ST/ST and EE/ST mice over the 8 wk postweaning. The
bar indicates the rearing condition over time (two-way ANOVA: rearing ef-
fect, P < 0.01; time effect, P < 0.0001; interaction, P < 0.0001. Bonferroni post
hoc tests for ST/ST vs. EE/ST mice, *P < 0.05; ***P < 0.001. Bonferroni post hoc
test applied to time course comparison for EE/ST,§§§P < 0.001). (D) Plasma
leptin at the end of the EE condition (P49) and 3 d after moving to standard
conditions (P53) in EE/ST and ST/ST mice (two-way ANOVA: time effect, P <
0.05; rearing effect, P < 0.01. Bonferroni post hoc test in EE/ST vs. ST/ST mice,
*P < 0.05. Bonferroni post hoc test applied to P49 vs. P53,§P < 0.05).
How EE experienced in youth affects food intake and metabolic
| www.pnas.org/cgi/doi/10.1073/pnas.0911832107 Mainardi et al.
Our results revealed that exposing mice to EE has an impact on
their feeding behavior, leptin production/action, and insulin
sensitivity. The former two outcomes depend on the age at which
EE is experienced, because EE in adulthood does not seem to
affect the leptin system, whereas EE since birth results in an en-
hanced response to exogenous leptin superior to that found in
physically exercised mice.
The ARC of young EE mice also exhibits important changes in
synaptic connectivity, with an increased excitation/inhibition ra-
tio in α-MSH neurons and a decreased excitation/inhibition ratio
in AgRP neurons.
Response to EE in Adult Mice. Although food intake is known to be
regulated by numerous factors and molecular pathways, the re-
sponse of adult mice to EE is largely predictable using the leptin
controller systemasa paradigm.Indeed, this condition resulted in
a reduction of fat mass, a consequent reduction of leptin pro-
duction, and increased food intake. The observed scenario mir-
rors the introduction of a more dynamic lifestyle in sedentary
individuals: Despite an augmented appetite, BW is maintained
well, with a beneficial effect on insulin sensitivity.
EE Since Birth Affects Leptin Action/Production. When compared
with YST mice, YEE mice exhibited similar fat depots, reduced
plasma leptin, and similar food intake, thus suggesting that the
leptin controller system had been adjusted to a different set point
with augmented sensitivity. Indeed, leptin response in YEE mice
in terms of food intake was more pronounced than in YST mice.
Consistently, YEE showed increased Ob-Rb hypothalamic ex-
pression and an enhanced response to leptin injection as assessed
by ARC STAT3 phosphorylation and food intake reduction. In-
creased expression of leptin receptor may well explain the
empowered leptin signaling, and STAT3 is considered the main
effector of leptin signaling in the ARC (6, 19). YEE mice also
showed increased hypothalamic expression of orexin. Despite the
positive effect on food intake described for this peptide, orexin-
deficient mice exhibit obesity, indicating that orexin may exert an
overall catabolic influence over energy balance (20).
Mice reared in EE since birth thus constitute a nongenetic and
nonpathological model of enhanced leptin sensitivity. Genetic
models with enhanced leptin sensitivity/response include, among
others, mice deficient for the cytokine signaling 3 (SOCS3) gene
(14, 21) and mice in which the orexin-OXR2 receptor signaling is
Comparison Between EE and Physical Exercise Effects on Metabolic
and Leptin Action/Production Outcomes. Patterson et al. (8) recently
reported that leptin resistance is reduced in diet-induced obese
rats exposed to postweaning voluntary exercise as compared
with control sedentary rats, thus attenuating the development of
obesity typical of this model. These data are in line with our
findings, although obtained in a pathological model genetically
predisposed to become obese and characterized by leptin re-
sistance (23). Indeed, the increase in leptin sensitivity herein
obtained for YEE mice indicates the possibility of manipulating
this parameter even in physiological conditions, as long as this
manipulation is applied early in life. In addition, our data un-
derscore three important differences between the effects of EE
and voluntary physical exercise. First, the effects of locomotor
activity on leptin sensitivity were less pronounced than those
observed in YEE mice. Second, some of the features in the YW
mice (reduced adiposity and increased food intake) totally dif-
fered from what found in YEE mice and cannot be interpreted
following the intermediate phenotype paradigm, thus suggesting
that physical exercise outcomes are not totally contained within
the EE condition and vice versa. Third, physical exercise did not
modify the overall density of excitatory or inhibitory synaptic
contacts in the ARC, whereas EE increases the former and
reduces the latter. Thus, components of EE other than physical
exercise could play a role in regulating feeding behavior and the
related mechanisms in developing mice. If we picture this concept
as a Venn diagram, we could say that the two conditions share an
intersection but maintain distinct areas of effect. In this regard,
evidence exists that EE and voluntary exercise affect different
phases of the neurogenic process in the hippocampus (24).
Trace Left by EE on the Leptin System on Treatment Discontinuation.
for 1 mo, the enhancement in leptin sensitivity disappeared but
the hypothalamic structural modifications persisted to some ex-
experienced during development.
Interestingly, food intake in EE/ST mice increased significantly
when they were placed in a standard cage, and a concomitant rise
in leptin was determined. These results suggest that the improved
leptin sensitivity observed in the YEE mice suddenly dropped on
environmental change; consequently, the hypothalamus sensed
leptin concentrations as abnormally low and animals were in-
duced to compensate for an imbalance in their leptin controller
system by eating more.
It is tempting to speculate about the existence of a leptin in-
hibitory signal promoted by EE and rapidly down-regulated on its
that informs the center (hypothalamus) about the status of the
energy stores (WAT). This putative leptin modulator would
complement leptin action and inform the periphery (WAT) about
the status of the central nervous system, the privileged target of
EE and the integration center of body functions. The nature of
thissignal, which wecan imagine as a circulating factor or nervous
input, has yet to be established. Of note, recent data (25) indicate
clear anatomical connections between POMC neurons in the
ARC and WAT.
and AgRP Neurons. In young mice, EE modified the excitatory/in-
hibitory synaptic connectivity in the ARC overall. Specifically,
α-MSH and AgRP neurons, respectively, displayed an enhance-
plasticity and changes in feeding behavior/energy homeostasis
were first linked by Pinto et al. (4), who found changes in the ex-
citation/inhibition of POMC and NPY cells of the ob/ob mouse,
consistent with the typical overfeeding behavior of this model.
ob ARC phenotype and that treatment of WT animals with the
orexigenic peptide ghrelin led to enhanced inhibition of POMC
neurons. Nutritional state has also been implicated in the rapid
reorganization of ARC synaptic connectivity. Studies in non-
human primates (26) indicate that fasting results in an altered
EE/ST mice. (A) Histogram showing the increase in the ratio between excit-
atory and inhibitory synapses on α-MSH neurons in the ARC of EE/ST (n = 4)
and ST/ST (n = 4) mice (t test, **P < 0.01). (B) Histogram showing the de-
crease in the ratio between excitatory and inhibitory synapses on AgRP
neurons in the ARC of EE/ST (t test, *P < 0.05).
Excitation/inhibition ratio on α-MSH and AgRP neurons of ST/ST and
Mainardi et al. PNAS
| September 21, 2010
| vol. 107
| no. 38
synaptic balance favoring the activity of NPY and orexin neurons.
hypothalamicareatoARCPOMC neurons wasreduced byfasting
in mice (16). In female mice, estradiol (E2) triggers a robust in-
crease in the number of excitatory inputs to POMC neurons in the
ARC that is independent of leptin (27). All changes so far de-
scribed resulted from a real (fasting) or perceived state of starva-
tion (ob/ob mouse) or following hormonal treatment.
Our studies provide a demonstration that a change in rearing
conditions and “lifestyle” may interfere with the neural circuitry
on ARC synapticconnectivity that leads totheopposite functional
outcome seen on ghrelin treatment, starvation, and leptin de-
ficiency. The enhanced leptin sensitivity herein observed in EE
mice could mediate the effects of EE on ARC synaptology; how-
ever, we cannot exclude the possibility that the observed changes
are independent of leptin but dependent on STAT3, as previously
demonstrated for E2 (27). In fact, enhanced basal activation of
STAT3 was observed in YEE mice as compared with YST mice.
Our results are in agreement with the observation that the
effect of EE on visual cortex plasticity involves a modulation of
the excitation/inhibition balance (18).
Overall, these findings indicate that diet-independent changes
in lifestyle occurring early in life are able to modulate leptin
sensitivity and leave metabolic and synaptic imprints. This may
have important implications for the use of leptin and behavioral
therapy in treatment, and especially for the prevention of obesity.
Materials and Methods
Detailed protocols are presented in SI Materials and Methods.
born in the EE condition and killed at P50, (ii) mice reared in the EE condition
since birth and then transferred to a standard cage at P50andmonitoreduntil
P80, (iii) adult mice moved to theEE conditionat P50andkilled at P80, and(iv)
mice reared since birth with free access to a running wheel and killed at P50.
Age-matched mice reared under standard conditions were used as controls.
Experimental protocols followed the Principles of Laboratory Animal Care
(authorization no. 129/2000-A from the Italian Ministry of Health).
Plasma Assays. Commercial ELISA kits were used to assess plasma levels of
insulin (Linco Research) and leptin (R&D Systems). Plasma glucose was
measured with a One-Touch Ultra glucometer (LifeScan).
IpGTT and AUC Parameters. IpGTTs were performed as described by Funicello
et al. (28). The total AUC was calculated using the trapezoid model.
Acute Leptin Injection. Animals were injected i.p. with saline or murine leptin
(3 mg/kg; Sigma) after a 1-h fast at dark onset. Food intake of the individually
caged animals was monitored 14 h after injection.
Isolation of Total RNA and Real-Time PCR. TotalRNAisolation,first-strandcDNA
relative abundance of mRNAs for ObRb, NPY, POMC, orexin, and BDNF was
calculated with TATA binding protein mRNA as an invariant control.
Immunofluorescence. After blocking, free-floating, 40-μm-thick, fixed-tissue
coronal sections were incubated with 1:1,000 rabbit anti-vGAT or -vGluT2,
anti-α-MSH antibodies. Sections were revealed with secondary antibodies
conjugated to Alexa-568 and Alexa-488.
Quantitative Analysis of Immunolabeled Cells and Puncta. Images were ac-
quired with a confocal laser-scanning microscope. For pSTAT3, STAT3, and
double-immunofluorescence images, the number of immunoreactive cells
and synaptic puncta was manually counted using MetaMorph software
(Universal Imaging Corp.). For pSTAT3 and STAT3 quantification, cell counts
were normalized to the ARC area in each section. For vGAT and vGluT-2
analysis, the images were processed using Imaris software (Bitplane).
Statistical Analysis. The number of mice in each experimental group is in-
dicated in the figure legends. All values are expressed as the mean ± SEM,
and a two-tailed Student’s t test was used for pairwise comparisons. One-
way and two-way ANOVA, followed by a Bonferroni post hoc test, was used
to compare more than two groups. Statistical evaluation was performed
using GraphPad Prism 3 (GraphPad Software).
ACKNOWLEDGMENTS. We thank Cesare Cocuzza for technical help in
metabolic studies. M. Maffei is an Associate Telethon Scientist. This work
was supported by Telethon Foundation Grants TCP99016 (to M. Maffei)
and GGP05236 (to T.P.), by Compagnia di San Paolo (Grant 2006-2008 to
Dulbecco Telethon Institute) and by the Italian Ministry of Health.
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| www.pnas.org/cgi/doi/10.1073/pnas.0911832107 Mainardi et al.