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The adipocyte hormone leptin sets the emergence of hippocampal inhibition in mice

  1. Camille Dumon
  2. Diabe Diabira
  3. Ilona Chudotvorova
  4. Francesca Bader
  5. Semra Sahin
  6. Jinwei Zhang
  7. Christophe Porcher
  8. Gary Wayman
  9. Igor Medina
  10. Jean-Luc Gaiarsa  Is a corresponding author
  1. Aix-Marseille University UMR 1249, INSERM (Institut National de la Santé et de la Recherche Médicale) Unité 1249, INMED (Institut de Neurobiologie de la Méditerranée), France
  2. Plateforme Post-Génomique, INMED, France
  3. Washington State University, United States
  4. University of Exeter Medical School, United Kingdom
Research Article
Cite this article as: eLife 2018;7:e36726 doi: 10.7554/eLife.36726
4 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
Early GABA developmental sequence in leptin-receptor deficient mice.

(A) Cell attached recordings of CA3 pyramidal neurons on acute hippocampal slices. Scale bar, 2 min, 50 pA. Corresponding time course of spike frequency changes are shown under each trace. (B) Developmental changes of isoguvacine action on spike activity. Mean ± SEM. (C) Current-voltage relationships for evoked GABAergic synaptic currents. Insets: examples of GABAergic synaptic current evoked at holding potentials ranging from −110 to −60 mV (10 mV increment) in db/db and from −70 to −30 (10 mV increment) in wt CA3 pyramidal neuron. Scale bar, 10 ms, 20 pA. (D) Developmental changes in EGABA and Em at zero current. Mean ± SEM. In B and D, the number of cells recorded and number of mice used are indicated in parenthesis; #p<0.05 when compared to pre-isoguvacine values, two-tailed paired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001 when compared to age-matched wt, two-tailed unpaired Student’s t-test. (E) Developmental profile of plasma leptin levels in the wt (blue circle) and developmental profile of ΔEGABA (gray circle). ΔEGABA was calculated as the difference in EGABA values between the wt and db/db at each developmental stage depicted in D. Numbers in parenthesis indicate the number of mice used. Mean +SEM. §p<0.05 when compared to P0 plasma leptin values, *p<0.05 and **p<0.01 when compared to P0 ΔEGABA values, one way ANOVA followed by a Tukey’s post hoc test. (F) Left: representative immuno-blots for hippocampal panKCC2 and β3-tubulin in wt and db/db mice (first two lanes). The third lane (-) illustrates background (empty well). Right: box plots of normalized pan KCC2 in P6 wt and db/db hippocampi. Numbers in parenthesis indicate the number of mice used. *p<0.05, two-tailed unpaired Student’s t-test.

https://doi.org/10.7554/eLife.36726.002
Figure 1—source data 1

Advanced onset of GABA inhibition in leptine receptor deficient mice.

https://doi.org/10.7554/eLife.36726.007
Figure 1—figure supplement 1
Leptin controls GABA signaling in the mice hippocampus in vivo.

(A) Plasma leptin levels in P8 wt, ob/ob and leptin-treated ob/ob mice (ob +lep, 5 mg/kg twice a day sub-cutaneous from P3 to P8, n = 4, blood samples were collected 30 min after the last injection). (B) Box plot of isoguvacine action on spike frequency in wt mice, ob/ and leptin-treated ob/ob mice at P8. (C,D) Box plots of EGABA and Em at zero current in 4 wt mice, ob/ob and leptin-treated ob/ob CA3 pyramidal neurons. The cells recorded and number of animal used are indicated in parenthesis *p<0.05, **p<0.01, one way ANOVA followed by a Tukey’s post hoc test.

https://doi.org/10.7554/eLife.36726.003
Figure 1—figure supplement 1—source data 1

Advanced onset of GABA inhibition in leptine deficient mice.

https://doi.org/10.7554/eLife.36726.004
Figure 1—figure supplement 2
KCC2 expression in db/db hippocampal neurons in vivo and leptin-treated hippocampal neuronal cultures in vitro.

(A) Confocal images showing KCC2 immuno-labeling in the CA3 pyramidal layer of wt and db/db hippocampi at P6. (B) Summary plot of the normalized KCC2 fluorescence intensity ratio in P6 wt and db/db hippocampal CA3 pyramidal layer. Calibration bar 20 µm. 3 wt and db/db littermate animals. mean ±sem. **p<0.01, two-tailed unpaired Student’s t-test. (C) Examples of KCC2 and MAP2 immunostaining in control and leptin-treated (100 nM, 24 hr) cultured hippocampal neurons (15 DIV). Scale bar 20 µM. (C) Summary plots of the normalized KCC2 fluorescence intensity in control (n = 30) and leptin-treated (n = 45) hippocampal neurons. Pooled data from 3 different cultures.

https://doi.org/10.7554/eLife.36726.005
Figure 1—figure supplement 3
Raw blots for panel F (WT and db).
https://doi.org/10.7554/eLife.36726.006
Leptin down-regulates KCC2 activity in cultured hippocampal neurons.

(A) I-V relationships for isoguvacine currents in vehicle (control) and leptin-treated (100 nM, 24 hr) hippocampal (15 DIV) cultures. Gramicidin perforated patch clamp recordings. Insets depict the isoguvacine currents. Scale bar, 500 ms, 100 pA. (B) Box plots of EGABA in the indicated conditions. *p<0.05, **p<0.01, ANOVA followed by a Tukey’s post hoc test. (C) Examples of isoguvacine currents (arrow heads) recorded at −80 and −40 mV before (t=-60 sec) and after (t = +20 and +420 s) neuronal chloride loading in control neuronal culture (15 DIV). Gramicidin perforated patch clamp recordings. Scale bar, 100 pA, 40 mV, 1 s. (D) Summary plots of normalized EGABA recovery after neuronal chloride loading in the indicated conditions. Mean ± SEM. In B and D, the number of cells recorded and number of cultures used are indicated in parenthesis.

https://doi.org/10.7554/eLife.36726.008
Figure 2—source data 1

Leptin decreases KCC2 activity in cultured hippocampal neurons.

https://doi.org/10.7554/eLife.36726.009
Leptin reduces the plasma membrane expression of KCC2 and modulates its phosphorylation state in cultured hippocampal neurons.

(A) Representative images illustrating total, membrane and internalized pools of KCC2 with external tag (KCC2-pHext) in vehicle and leptin-treated (100 mM, 24 hr) cultured hippocampal neurons expressing a scramble Sh-RNA (Sh-Sbl). Neurons expressing KCC2 with internal tag (KCC2-pHint) were proceeded in parallel experiments to ensure that immunocytochemistry on living neurons does not permeabilized the membrane. Scale bars 20 μm and 1 μm. (B) Box plots of normalized membrane (Fm) and internalized (Fi) fluorescence in vehicle and leptin-treated (+Lep) cultured neurons expressing the indicated constructs. *p<0.05, one way ANOVA followed by a Tukey’s post hoc test. (C) Western blots and quantifications (D and E) of KCC2, NKCC1, KCC2/NKCC1 ratio and the threonine 906, threonine 1007 and serine 940-phosphorylated forms of KCC2 in control and leptin (100 nM, 24 hr)-treated hippocampal neuronal cultures (DIV15, five independent neuronal cultures). ***p<0.001, two-tailed unpaired Student’s t-test. (F) Box plots of EGABA in the indicated conditions. Gramicidin perforated patch clamp recordings were performed on hippocampal neuronal cultures at 15 DIV. **p<0.01, ***p<0.001, one way ANOVA followed by a Tukey’s post hoc test. In B and F, the number of cells recorded and number of cultures used are indicated in parenthesis.

https://doi.org/10.7554/eLife.36726.010
Figure 3—source data 1

Leptine controls the membrane expression of KCC2 and its phosphorylated state in hippocampal culture via the WNK pathway.

https://doi.org/10.7554/eLife.36726.011
Figure 4 with 3 supplements
Hyperleptinemia and maternal obesity delay the GABA developmental sequence and downregulate KCC2 expression.

(A) Cell attached recordings of CA3 pyramidal neurons on acute hippocampal slices obtained from pups of normal diet (ND) and diet-induced obese (DIO) dams at P10. (B) Developmental changes of isoguvacine action on spike frequency. Mean +SEM. (C) Cell attached recordings of CA3 pyramidal neurons on acute hippocampal slices obtained from vehicle-treated (sham) and leptin-treated mice at P10. (D) Box plots of isoguvacine action on spike activity. In B and D, number of cells recorded and number of mice used are indicated in parenthesis; #p<0.05 when compared to pre-isoguvacine values, two-tailed paired Student’s t-test and *p<0.05 and **p<0.01 when compared to age matched ND-pups (B and C) or sham-pups (E), two-tailed unpaired Student’s t-test. (E) Representative immuno-blots for hippocampal panKCC2 and β3-tubulin in offspring of DIO and ND dams at P13 and in control (sham) and leptin-treated (Lep) mice at P10. The third lanes (-) illustrate background (empty wells). (F) Normalized panKCC2 immunoreactivity in ND (n = 6 pups) and sham (n = 3 pups), in offspring of DIO (n = 5 pups) and in leptin-treated mice (n = 5 pups). Mean +SEM. *p<0.05, two-tailed unpaired Student’s t-test.

https://doi.org/10.7554/eLife.36726.012
Figure 4—source data 1

Maternal obesity and hyperleptinemia delayed the emergence of functional GABAergic inhibition.

https://doi.org/10.7554/eLife.36726.016
Figure 4—figure supplement 1
High fat diet induced obesity.

(A) Experimental overview. (B) Body weight curves of adult (8 weeks old) female mice fed with normal diet (10% kcal from fat, ND white symbols, n = 4) or a high-fat diet (60% kcal from fat, HFD, n = 12). (C) Average body weight increase after 6 weeks of food supply. *p<0.05, **p<0.01, two-tailed unpaired Student’s t-test.

https://doi.org/10.7554/eLife.36726.013
Figure 4—figure supplement 2
Raw blots for panel E (ND and DIO).
https://doi.org/10.7554/eLife.36726.014
Figure 4—figure supplement 3
Raw blots for panel E (Lep and sham).
https://doi.org/10.7554/eLife.36726.015

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(M. musculus)
B6.Cg-Lepob/JThe Jackson
Laboratory
RRID:IMSR_JAX:000632
Genetic reagent
(M. musculus)
B6.BKS-LeprdbThe Jackson
Laboratory
RRID:IMSR_JAX:000697
Transfected
DNA construct
shLepR 3426PMID:24877561,
DOI: 10.1210/me.2013–1332
Dr. G. Wayman
(Washington
State University)
Transfected
DNA construct
shLepR 3299PMID:24877561,
DOI: 10.1210/me.2013–1332
Dr. G. Wayman
(Washington
State University)
Transfected
DNA construct
WNK shRNAPMID:
26126716, DOI: 10.1126
/scisignal.aaa0354
Transfected
DNA construct
WNK-CAPMID:
26126716, DOI: 10.1126
/scisignal.aaa0354
Transfected
DNA construct
KCC2-pHluorinPMID:
24928908, DOI: 10.15252
/embr.201438840
AntibodyMouse anti-β3 tubulinSigma-AldrichRRID:AB_477590,
Cat# T8660
WB (1:10 000)
AntibodyRabbit anti-KCC2US BiologicalRRID:AB_2188802,
Cat# K0120-07
WB (1:2000)
AntibodyDonkey anti-chiken Alexa488FluoprobesRRID: AB_2686906,
Cat# FP-SA5110
IHC (1:1000)
AntibodyChicken anti-MAP2AbcamRRID:AB_2138153,
Cat# ab5392
IHC (1:2000)
AntibodyDonkey Anti-rabbit Cy3ChemiconRRID:AB_92588,
Cat# AP182C
IHC (1:1000)
Antibodymouse Anti-GFPNovus BiologicalsRRID:AB_531011,
Cat# NB 600–597
AntibodyNKCC1 totalthe Division of Signal
Transduction Therapy
Unit (DSTT) at the
University of Dundee
dundee (S022D)WB (1 mg/ml)
Antibodypan KCC2Dr. C. Rivera
(University of Helsinki)
AntibodyKCC2 Ser940Novus BiologicalsCat# NBP2-29513WB (1 mg/ml)
AntibodyKCC2a phosphoT1007the Division of Signal
Transduction Therapy
Unit (DSTT) at the
University of Dundee
dundee (S959C)WB (1 mg/ml)
AntibodyKCC2a phosphoT906the Division of Signal
Transduction Therapy
Unit(DSTT) at the
University of Dundee
dundee (S959C)WB (1 mg/ml)
OligonucleotidesSlc12a2 (NKCC1)QiagenQT00197785
OligonucleotidesSlc 12a5 (KCC2)QiagenQT00145327
OligonucleotidesGAPDHQiagenQT001199633
Peptide,
recombinant protein
Recombinant
murine leptin
Tocris,Cat# TO-2985/1
Commercial
assay or kit
Mouse leptin
ELISA kit
BioVendor R and DRCat# RD291001200
Chemical
compound,
drug
1,2,3,4-Tetrahydro-6
-nitro-2,3-dioxo-benzo
[f]quinoxaline-7-
sulfonamide (NBQX)
NIMH Chemical
Synthesis and Drug
Supply Program,
https://nimh-repository.rti.org/
Chemical
compound,
drug
D-2-amino-5-
phosphovaleric
acid (D-APV)
NIMH Chemical
Synthesis and Drug
Supply Program,
https://nimh-repository.rti.org/
Chemical
compound,
drug
1,2,3,4-Tetrahydro-6
-nitro-2,3-dioxo-benzo
[f]quinoxaline-7-
sulfonamide (NBQX)
NIMH Chemical
Synthesis and
Drug Supply
Program,
https://nimh-repository.rti.org/
Chemical
compound,
drug
IsoguvacineTocrisCat# 0235
Chemical
compound,
drug
VU0463271TocrisCat# 4719
Chemical
 compound,
drug
BumetanideSigmaCat# B3023
Software
and
Algorithms
National Institutes of HealthRRID:SCR_003070
Software
and
Algorithms
Synaptosoft, http://www.synaptosoft.com/MiniAnalysis/DownloadDemo.html

Data availability

All data generated or analyszed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 to 4 and Supplementary Figures 1 and 2.

Additional files

Supplementary file 1

Advanced onset of GABA inhibition in leptin deficient mice.

https://doi.org/10.7554/eLife.36726.017
Transparent reporting form
https://doi.org/10.7554/eLife.36726.018

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